KR20130086386A - Biosynthetically generated pyrroline-carboxy-lysine and site specific protein modifications via chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues - Google Patents

Biosynthetically generated pyrroline-carboxy-lysine and site specific protein modifications via chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues Download PDF

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KR20130086386A
KR20130086386A KR1020137017122A KR20137017122A KR20130086386A KR 20130086386 A KR20130086386 A KR 20130086386A KR 1020137017122 A KR1020137017122 A KR 1020137017122A KR 20137017122 A KR20137017122 A KR 20137017122A KR 20130086386 A KR20130086386 A KR 20130086386A
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pcl
formula
cells
protein
pyrrolysine
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KR1020137017122A
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Korean (ko)
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베른하드 가이어스탄거
웨이지아 오우
수잔 이. 셀리티
테쯔오 우노
티파니 크로스그로브
시엔-포 치우
얀 그루네발트
쉐시 하오
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아이알엠 엘엘씨
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Priority to US61/108,434 priority
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Priority to PCT/US2009/061954 priority patent/WO2010048582A1/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D207/00Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/18Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
    • C07D207/20Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D207/00Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C07D207/48Sulfur atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu

Abstract

Disclosed herein are pyrroline-carboxy-lysine (PCL), a biosynthetically produced natural amino acid and a pyrrolysine analog, and a method for biosynthetically producing PCL. Also disclosed herein are proteins, polypeptides and peptides having PCL incorporated therein, and methods of incorporating PCL into such proteins, polypeptides and peptides. Also disclosed herein are site specific derivatization of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein. Also disclosed herein are crosslinking of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein.

Description

Biosynthetically produced pyrroline-carboxy-lysine and site-specific protein modification via chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues DERIVATIZATION OF PYRROLINE-CARBOXY-LYSINE AND PYRROLYSINE RESIDUES}

<Cross reference to related application>

This application claims the benefit of priority to US Provisional Patent Application 61 / 108,434, filed October 24, 2008, under 35 U.S.C. §119 (e). The disclosure of this priority application is incorporated herein by reference in its entirety for all purposes.

<Technical Field>

The present invention relates to the selective introduction of genetically encoded amino acids into proteins. The invention also relates to chemical derivatization of such amino acids.

The methylamine methyltransferase of the methanogenic archaea of the family Methanosarcina naturally contains pyrrolysine (PYL). Pyrrolysine is a lysine analog that is incorporated at the same time as translation in the in-frame UAG codon in each mRNA, which is considered the 22nd natural amino acid.

Provided herein are proteins and / or polypeptides having one or more PCL incorporated therein, where the PCL is biosynthetically produced and incorporated into the protein and / or polypeptide. Also provided herein are proteins and / or polypeptides having one or more pyrrolysine (PYL) incorporated therein, wherein the PYL is biosynthetically produced and incorporated into the protein and / or polypeptide. Also provided herein are proteins and / or polypeptides having one or more PCL incorporated therein and one or more PYL incorporated therein, wherein the PCL and PYL are biosynthesically produced and incorporated into the protein and / or polypeptide. .

Also provided herein are proteins and / or polypeptides having one or more PCL residues, wherein the PCL is biosynthetically produced and incorporated into the protein and / or polypeptide, and the one or more PCL residues are labeled, dyes, polymers, Water-soluble polymers, polyalkylene glycols, poly (ethylene glycol), derivatives of poly (ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic residues, isotopically labeled residues, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent residues, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores, Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes, Activated complex activator, virus, toxin, adjuvant, TLR2 agonist, TLR4 agonist, TLR7 agonist, TLR9 agonist, TLR8 agonist, T-cell epitope, phospholipids, LPS-like molecule, keyhole limpet hemocyanin (KLH), immunogenic hapten, aglycans, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, mimotopes, receptors, reverse micelles, detergents, immune boosters, Neon Dyes, FRET reagents, radiation-imaging probes, other spectroscopic probes, prodrugs, immunotherapy toxins, solid supports, -CH 2 CH 2- (OCH 2 CH 2 O) p -OX 2 , and -O- (CH 2 CH 2 O) p CH 2 CH 2 -X 2 ( wherein, p is 1 to 10,000, X 2 is H, C 1 - coupling group selected from 8 alkyl, protecting group, or the terminal functional group Im) to a protein and / or polypeptide By means of ring derivatization.

In certain embodiments of such proteins and / or polypeptides having one or more PYL residues incorporated therein, wherein PYL is biosynthesically produced and incorporated into proteins and / or polypeptides, pyrrolysine is incorporated therein. For proteins and / or polypeptides having more than one PCL residue, one of the above-mentioned groups given above is derivatized thereby by coupling to the protein and / or polypeptide.

In certain embodiments of such proteins and / or polypeptides having one or more PCL and one or more PYL residues incorporated therein, wherein the PCL and PYL are biosynthesically produced and incorporated into proteins and / or polypeptides, the PCL And pyrrolysine is derivatized thereby by coupling one of the above-mentioned groups given above to a protein and / or polypeptide to a protein and / or polypeptide having one or more PCL residues incorporated therein.

In certain embodiments, such aforementioned biosynthesis occurs in eukaryotic cells, mammalian cells, yeast cells, or insect cells. In certain embodiments, the cells are Escherichia coli cells, while in other embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the cells are CHO cells, HeLa cells, HEK293F cells or sf9 cells.

One aspect provided herein is a compound having a structure of Formula I or Formula II below.

<Formula I>

Figure pat00001

&Lt;

Figure pat00002

Where

R 1 is H or an amino terminal modification group;

R 2 is OH or a carboxy terminus modification group;

n is an integer from 1 to 5000;

Each AA is independently selected from an amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-2, and a pyrrolysine analog amino acid residue having the structure of Formula B-2;

Each BB is an amino acid residue, a pyrrolysine analogue having the structure of Formula A-2 amino acid residue, a pyrrolysine analogue having the structure of Formula B-2, a pyrrolysine analogue having the structure of Amino acid residue, pyrrolysine analogue amino acid having a structure of formula D-1, pyrrolysine analog amino acid residue, having a structure of formula E-1, pyrrolysine analog amino acid residue, having a structure of formula F-1 A pyrrolysine analog amino acid residue having a structure of Formula G-1, a pyrrolysine analog amino acid residue having a structure of Formula H-1, a pyrrolysine analog amino acid residue having a structure of Formula I-1, A pyrrolysine analog amino acid residue having the structure of Formula J-1, a pyrrolysine analog amino acid residue having the structure of Formula K-1, and a structure of Formula L-1 It is independently selected from pyrrolidin new analogues of amino acid residues;

Figure pat00003

Where

R 3, R 5 and each R 4 is H, -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

R 6 is H or C 1 alkyl;

A is C 3 -C 8 cycloalkyl, C 3 -C 8 heterocycloalkyl, 5 to 6 membered monocyclic aryl, 5 to 6 membered monocyclic heteroaryl, fused 9 to 10 membered bicyclic ring or fused 13 a to 14-membered tricyclic ring, where A is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene, -O (CR 11 R 12) k -, -S (CR 11 R 12) k -, -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C ( O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11- , -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k- , -C (O) NR 11 (CR 11 R 12) k -, -NR 11 C (O) (CR 11 R 12) k - is selected from wherein each R 11 and R 12 are independently H, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl or hydroxy-substituted -C 1 - 8 is alkyl, k is an integer from 1 to 12;

X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X is selected from the second and any combination thereof, wherein p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl, protecting group or a terminal functional group;

At least one AA is a pyrrolysine analog amino acid residue having the structure of Formula A-2 or Formula B-2, or at least one BB is Formula C-1 or Formula D-1 or Formula E-1 or Formula F- Or pyrrolysine analog amino acid residue having a structure of Formula G-1 or Formula H-1 or Formula I-1 or Formula J-1 or Formula K-1 or Formula L-1.

In certain embodiments of said compounds, Ring A is furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyra Sleepy, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine , Pyrazine, piperazine, triazine, trithiane, indoliazine, indole, isoindole, indolin, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolyzine, quinoline, iso Quinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidin, carbazole, acridine, phenazine, pentazine, phenoxazine, phenyl, indene, naphthalene, azulene , Fluorene, anthracene, phenanthracene, norborane and adamantin.

In another embodiment of this compound, Ring A is phenyl, furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole , Pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, Pyrimidine, pyrazine, piperazine, triazine and trithiane.

In another embodiment of this compound, Ring A is indolizin, indole, isoindole, indolin, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolyzine, quinoline, iso Quinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidin, carbazole, acridine, phenazine, pentazine, phenoxazine, indene, naphthalene, azulene, flu Orene, anthracene, phenanthracene, norborane and adamantin.

In certain embodiments of the compound, Ring A is selected from phenyl, naphthalene and pyridine.

In certain embodiments of the compounds, each BB is an amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-2, a pyrrolysine analog amino acid residue having the structure of Formula B-2, Formula C Pyrrolysine analog amino acid residue having a structure of -1, pyrrolysine analog amino acid residue having a structure of formula D-2, pyrrolysine analog amino acid residue having a structure of formula E-1, formula F-2 Pyrrolysine analog amino acid residue having the structure of, Pyrrolysine analog amino acid residue having the structure of Formula G-1, Pyrrolysine analog amino acid residue having the structure of Formula H-2, Structure of formula I-1 Pyrrolysine analogue amino acid residue having, pyrrolysine analogue having structure of formula J-2, pyrrolysine analogue having structure of formula K-1 Amino acid residue and the following independently selected from pyrrolidin new analogues of amino acid residues having the structure of Formula L-2.

Figure pat00004

Where

R 3, R 5 and each R 4 is H, -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

R 6 is H or C 1 alkyl;

When present, each R 7 is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, aryl, heteroaryl Independently heterocycloalkyl or cycloalkyl, and -LX 1 ;

L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene, polyalkylene glycols, poly (ethylene glycol), -O (CR 11 R 12 ) k -, -S (CR 11 R 12) k - , -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C (O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11 -, -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k- , -C (O) NR 11 (CR 11 R 12 ) k- , -NR 11 C (O) (CR 11 R 12 ) k- , wherein each of R 11 and R 12 is independently H, C 1 - 8 alkyl, halo-substituted -C 1-8 alkyl, or hydroxy-substituted -C 1 - 8 is alkyl, k is an integer from 1 to 12;

X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X is selected from the second and any combination thereof, wherein p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl, protecting group or a terminal functional group.

In certain embodiments of the compound, R 6 is H, while in other embodiments of the compound, R 6 is C 1 alkyl.

In certain embodiments of the compound, R 5 is -LX 1 . In certain embodiments of the compound, R 7 is -LX 1 . In certain embodiments of the compounds, X 1 is a sugar, polyethylene glycol, fluorescent moiety, immunomodulator, ribonucleic acid, deoxyribonucleic acid, protein, peptide, biotin, phospholipid, TLR7 agonist, immunogenic hapten or solid support. In certain embodiments of the above compounds, L is a poly (alkylene glycol), poly (ethylene glycol), C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene or hydroxy-substituted -C 1 8 is an alkylene group.

Another aspect provided herein is a method of derivatizing a protein comprising contacting a protein having a structure according to Formula I with a reagent of Formula III or Formula IV below.

<Formula I>

Figure pat00005

Where

R 1 is H or an amino terminal modification group;

R 2 is OH or a carboxy terminus modification group;

n is an integer from 1 to 5000;

Each AA is independently selected from an amino acid residue, a pyrrolysine amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-1, and a pyrrolysine analog amino acid residue having the structure of Formula B-1, ;

Figure pat00006

R 6 is H or C 1 alkyl;

At least one AA is a pyrrolysine amino acid residue or a pyrrolysine analog amino acid residue having a structure of Formula A-1 or Formula B-1.

<Formula III>

Figure pat00007

(IV)

Figure pat00008

Where

R 3, R 5 and each R 4 is H, -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

A is C 3 -C 8 cycloalkyl, C 3 -C 8 heterocycloalkyl, 5 to 6 membered monocyclic aryl, 5 to 6 membered monocyclic heteroaryl, fused 9 to 10 membered bicyclic ring or fused 13 a to 14-membered tricyclic ring, where A is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene group, a poly (alkylene glycol), poly (ethylene glycol), -O (CR 11 R 12 ) k -, -S (CR 11 R 12) k- , -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C (O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11- , -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k −, —C (O) NR 11 (CR 11 R 12 ) k −, —NR 11 C (O) (CR 11 R 12 ) k- , wherein each R 11 and R 12 is independently H , C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl or hydroxy-substituted -C 1 - 8 is alkyl, k is an integer from 1 to 12;

X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X is selected from the second and any combination thereof, wherein p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl, protecting group or a terminal functional group.

In certain embodiments of the aforementioned methods, the amino acid residues of formula A-1 are amino acid residues having the structure of formula A-2 or formula A-3.

Figure pat00009

In certain embodiments of the aforementioned methods, the amino acid residues of formula B-1 are amino acid residues having the structure of formula B-2 or formula B-3.

Figure pat00010

In certain embodiments of the aforementioned methods, the amino acid residues of formula A-1 are residues of amino acids of formula V and the amino acid residues of formula B-1 are residues of amino acids of formula VI.

Figure pat00011

Wherein R 6 is H or C 1 alkyl

In certain embodiments, the amino acids of Formula (V) or Formula (VI) are biosynthetically produced in a cell comprising a pylB gene, pylC gene, and pylD gene, and the cell is in contact with a growth medium comprising a precursor. In other embodiments, the amino acids of Formula V or Formula VI are biosynthetically produced in a cell comprising a pylC gene and a pylD gene, and the cells are in contact with a growth medium comprising a precursor.

In certain embodiments, the amino acid of Formula (V) is an amino acid having the structure of Formula (VII) wherein the precursor is ornithine, arginine, D-ornithine, D-arginine, (2S) -2-amino-6- (2,5 -Diaminopentaneamido) hexanoic acid or (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.

Figure pat00012

In certain embodiments, the amino acid of Formula VI is an amino acid having the structure of Formula VIII wherein the precursor is ornithine or arginine, and the precursor is ornithine, arginine, D-ornithine, D-arginine, (2S) -2- Amino-6- (2,5-diaminopentaneamido) hexanoic acid or (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.

Figure pat00013

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula VII, and the precursor is D-ornithine or D-arginine. In certain embodiments, the amino acid of Formula VI is an amino acid having the structure of Formula VIII, and the precursor is D-ornithine or D-arginine. In certain embodiments, the amino acid of formula V is an amino acid having the structure of formula VII, and the precursor is (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the amino acid of formula V is an amino acid having the structure of formula VII, and the precursor is (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the amino acid of formula VI is an amino acid having the structure of formula VIII, and the precursor is (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the amino acid of formula VI is an amino acid having the structure of formula VIII, and the precursor is (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is ornithine, arginine, D-ornithine, D-arginine or 2,5-diamino-3-methylpentanoic acid.

Figure pat00014

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X, and the precursor is ornithine, arginine, D-ornithine, D-arginine or 2,5-diamino-3-methylpentanoic acid.

Figure pat00015

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is D-2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of formula V is an amino acid having the structure of formula X and the precursor is D-2,5-diamino-3-methylpentanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is (2R, 3S) -2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X and the precursor is (2R, 3S) -2,5-diamino-3-methylpentanoic acid.

In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula IX and the precursor is (2R, 3R) -2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X, and the precursor is (2R, 3R) -2,5-diamino-3-methylpentanoic acid. In certain embodiments, the amino acid of formula V is an amino acid having the structure of formula IX, and the precursor is D-ornithine or D-arginine or (2S) -2-amino-6-((R) -2,5-di Aminopentaneamido) hexanoic acid. In certain embodiments, the amino acid of Formula V is an amino acid having the structure of Formula X, and the precursor is D-ornithine or D-arginine or (2S) -2-amino-6-((R) -2,5-di Aminopentaneamido) hexanoic acid.

In certain embodiments of the aforementioned methods, the amino acids of Formula (V), Formula (VI), Formula (VII), Formula (VII), Formula (IX), or Formula (X) are directed to orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS). Incorporated into intracellular proteins, where the O-RS aminoacylates the O-tRNA with an amino acid of Formula V or Formula VI, and the O-tRNA recognizes one or more selector codons of mRNA in the cell.

In certain embodiments of the aforementioned methods, the cell further comprises a pylS gene and a pylT gene, wherein the amino acid of Formula V, Formula VI, Formula VII, Formula VII, Formula IX or Formula X is an aminoacyl tRNA synthetase, and The cells are incorporated into intracellular proteins by tRNAs that recognize one or more selector codons of mRNA, wherein the aminoacyl tRNA synthetase is the gene product of the pylS gene and the tRNA is the gene product of the pylT gene.

In certain embodiments of the aforementioned method, the selector codon is an amber codon (TAG).

In certain embodiments of the aforementioned methods, the cells are prokaryotic cells, while in other embodiments the cells are eukaryotic cells. In certain embodiments, the cells are Escherichia coli cells, while in other embodiments the cells are mammalian cells, yeast cells, or insect cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cell is a CHO cell, HeLa cell or HEK293F cell. In certain embodiments, the insect cell is an sf9 cell.

Another aspect provided herein is a derivatized protein obtained using the above-mentioned method, wherein such derivatized protein has a structure according to formula II.

&Lt;

Figure pat00016

Where

R 1 is H or an amino terminal modification group;

R 2 is OH or a carboxy terminus modification group;

n is an integer from 1 to 5000;

Each BB is an amino acid residue, a pyrrolysine analogue amino acid having a structure of Formula A-1, a pyrrolysine analog amino acid residue having a structure of Formula B-1, a pyrrolyl having a structure of Formula C-1 New analogue amino acid residues, pyrrolysine analogues having the structure of Formula D-1. New analogue amino acid residues, pyrrolysine analogues having the structure of Formula E-1. Amino acid residue, pyrrolysine analogue amino acid having a structure of formula G-1, pyrrolysine analog amino acid residue having a structure of formula H-1, pyrrolysine analog amino acid residue of structure of formula I-1 , A pyrrolysine analog amino acid residue having the structure of Formula J-1, a pyrrolysine analog amino acid residue having the structure of Formula K-1, and Blood tank having a Raleigh new analogues are independently selected from amino acid residues;

Figure pat00017

One or more BBs are represented by Formula C-1 or Formula D-1 or Formula E-1 or Formula F-1 or Formula G-1 or Formula H-1 or Formula I-1 or Formula J-1 or Formula K-1 or Pyrrolysine analog amino acid residue having the structure of Formula L-1.

In certain embodiments of the aforementioned methods and said derivatized proteins, Ring A is furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imida Zolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomor Pauline, pyridazine, pyrimidine, pyrazine, piperazine, triazine, trithiane, indolinazine, indole, isoindole, indolin, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, Quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinoclidine, carbazole, acridine, phenazine, pentazine, phenoxazine, phenyl From indene, naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantin It is selected.

In certain embodiments of the aforementioned methods and said derivatized proteins, Ring A is phenyl, furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, Imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, Thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine and trithiane.

In certain embodiments of the above-mentioned methods and said derivatized proteins, ring A is indolizin, indole, isoindole, indolin, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quino Teasing, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidin, carbazole, acridine, phenazine, pentazine, phenoxazine, indene, Naphthalene, azulene, fluorene, anthracene, phenanthracene, norborane and adamantine.

In certain embodiments of the aforementioned methods and the derivatized protein, Ring A is selected from phenyl, naphthalyl and pyridyl.

In certain embodiments of the aforementioned methods each BB is an amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-2, a pyrrolysine analog amino acid residue having the structure of Formula B-2, Pyrrolysine analog amino acid residue having a structure of C-1, Pyrrolysine analog amino acid residue having a structure of Formula D-2, Pyrrolysine analog amino acid residue having a structure of Formula E-1, Formula F- Pyrrolysine analog amino acid residue having a structure of 2, pyrrolysine analog amino acid residue having a structure of Formula G-1, pyrrolysine analog amino acid residue having a structure of Formula H-2, Pyrrolysine analog amino acid residue having a structure, pyrrolysine analog amino acid residue having a structure of Formula J-2, pyrrolysine analogous structure having the structure of Formula K-1 Body amino acid residues and pyrrolysine analog amino acid residues having the structure of Formula L-2:

Figure pat00018

Where

R 3, R 5 and each R 4 is H, -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

R 6 is H or C 1 alkyl;

When present, each R 7 is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, aryl, heteroaryl Independently heterocycloalkyl or cycloalkyl, and -LX 1 ;

L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene, polyalkylene glycols, poly (ethylene glycol), -O (CR 11 R 12 ) k -, -S (CR 11 R 12) k - , -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C (O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11 -, -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k- , -C (O) NR 11 (CR 11 R 12 ) k- , -NR 11 C (O) (CR 11 R 12 ) k- , wherein each of R 11 and R 12 is independently H, C 1 and 8, and the alkyl, k is an integer from 1 to 12, - 8 alkyl, halo-substituted -C 1 - 8 alkyl or hydroxy-substituted -C 1

X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X is selected from the second and any combination thereof, wherein p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl, protecting group or a terminal functional group.

In certain embodiments of the aforementioned methods and said derivatized protein, R 6 is H, while in other embodiments R 6 is C 1 alkyl.

In certain of the aforementioned methods and in certain derivatized proteins, R 5 is -LX 1 . In certain embodiments of the above-mentioned methods and said derivatized proteins, X 1 is a sugar, polyethylene glycol, fluorescent moiety, immunomodulator, ribonucleic acid, deoxyribonucleic acid, protein, peptide, biotin, phospholipid, TLR7 agonist, immune Original hapten or solid support. In certain embodiments, L is a poly (alkylene glycol), poly (ethylene glycol), C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene or hydroxy-substituted -C 1 - 8 alkyl, Ren.

In certain embodiments of the aforementioned methods, the reagent of formula IV is

Figure pat00019
Wherein L and X 1 are as described above.

In certain embodiments of such reagents, L is a bond and X 1 is polyethylene glycol.

In certain embodiments, the reagent of Formula IV is

Figure pat00020

Figure pat00021

Figure pat00022

Wherein the compound having one or more polyethylene glycol (PEG) residues has an average molecular weight in the range of 1000 Da to 50 kDa, n is 20 to 1200 and exPADRE is

Figure pat00023
And PADRE is
Figure pat00024
And BG1 is
Figure pat00025
And BG2 is
Figure pat00026
Where * represents a phosphothioate linkage.

In certain embodiments, the reagent of Formula IV is of the structure:

Figure pat00027
Wherein the compound has an average molecular weight in the range of 1000 Da to 30 kDa and n is 20 to 679.

In another embodiment, the reagent of Formula (IV) has the structure

Figure pat00028
Wherein the compound has an average molecular weight in the range of 1000 Da to 45 kDa and n is 20 to 1018.

Another aspect provided herein is a compound having a structure of Formula VII or Formula VIII wherein the compound of Formula VII or Formula VIII is biosynthetically produced in a cell comprising a pylC gene and a pylD gene, and the cell is a precursor It is in contact with a growth medium comprising a.

(VII)

Figure pat00029

&Lt; Formula (VIII)

Figure pat00030

In certain embodiments of the compounds, the cell comprises a pylB gene, pylC gene and pylD gene.

In certain embodiments of the compounds, the precursor is ornithine or arginine, while in other embodiments the precursor is D-ornithine or D-arginine or (2S) -2-amino-6-((R) -2,5 Diaminopentaneamido) hexanoic acid.

In certain embodiments of the compounds, compounds of Formula (VII) or Formula (VIII) are incorporated into intracellular proteins by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is O -tRNA is aminoacylated with a compound of Formula V or Formula VI, and the O-tRNA recognizes one or more selector codons of mRNA in the cell.

In certain embodiments of the compound, the cell further comprises a pylS gene and a pylT gene, wherein the compound of Formula V or Formula VI is comprised by an aminoacyl tRNA synthetase and a tRNA that recognizes one or more selector codons of mRNA in the cell. Incorporated into intracellular proteins, wherein the aminoacyl tRNA synthetase is the gene product of the pylS gene and tRNA is the gene product of the pylT gene.

In certain embodiments the selector codon is an amber codon (TAG). In certain embodiments, the cells are prokaryotic cells, while in other embodiments the cells are eukaryotic cells. In certain embodiments, the cells are Escherichia coli cells, while in other embodiments the cells are mammalian cells, yeast cells, or insect cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cell is a CHO cell, HeLa cell or HEK293F cell. In certain embodiments, the insect cell is an sf9 cell.

Another aspect provided herein is a compound having a structure of Formula VII or Formula VIII, wherein the compound of Formula VII or Formula VIII is biosynthesized by a first cell in contact with a growth medium comprising a precursor and a second cell Are produced and secreted, wherein the first cell is a feeder cell comprising a pylC gene and a pylD gene.

(VII)

Figure pat00031

&Lt; Formula (VIII)

Figure pat00032

In certain embodiments of the compounds, the first cell comprises a pylB gene, pylC gene and pylD gene.

In certain embodiments of such compounds, the precursor is ornithine or arginine. In other embodiments of the compounds, the precursor is D-ornithine or D-arginine. In another embodiment of the compound, the precursor is (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid. In another embodiment of the compound, the precursor is (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.

In certain embodiments of the compounds, the compound of Formula VII or Formula VIII is incorporated into the second intracellular protein by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS Amino-acylates the O-tRNA with a compound of Formula VII or Formula VIII, and the O-tRNA recognizes one or more selector codons of mRNA in the second cell.

In certain embodiments of the compound, the second cell comprises a pylS gene and a pylT gene, and the compound of Formula VII or Formula VIII is incorporated into a protein in the second cell.

In certain embodiments of such compounds, a compound of Formula VII or Formula VIII is incorporated into a protein in a second cell by an aminoacyl tRNA synthetase, and a tRNA that recognizes one or more selector codons of mRNA in the cell, wherein the aminoacyl tRNA Synthetase is the gene product of the pylS gene and tRNA is the gene product of the pylT gene.

In certain embodiments of the compounds, the selector codon is an amber codon (TAG).

In certain embodiments of the compounds, the first cell or the second cell is a prokaryotic cell. In certain embodiments of the compounds, the first and second cells are prokaryotic cells. In certain embodiments of the compounds, the first cell or the second cell is a eukaryotic cell. In certain embodiments of the compounds, the first cell and the second cell are eukaryotic cells.

In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments, the eukaryotic cells are mammalian cells, yeast cells or insect cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cell is a CHO cell, HeLa cell or HEK293F cell. In certain embodiments, the insect cell is an sf9 cell.

Another aspect provided herein is a compound having a structure of Formula IX, wherein the compound of Formula IX is biosynthetically produced in a cell comprising a pylC gene and a pylD gene, wherein the cell is a 2,5-diamino- Contact with a growth medium comprising 3-methylpentanoic acid or D-2,5-diamino-3-methylpentanoic acid.

<Formula IX>

Figure pat00033

In certain embodiments of the compounds, 2,5-diamino-3-methylpentanoic acid is (2R, 3S) -2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the compounds, 2,5-diamino-3-methylpentanoic acid is (2R, 3R) -2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the compounds, the cell comprises a pylB gene, pylC gene and pylD gene, and the cell is ornithine, arginine, D-ornithine, D-arginine, (2S) -2-amino-6- (2 , 5-diaminopentaneamido) hexanoic acid is contacted with a growth medium comprising 2,5-diamino-3-methylpentanoic acid or D-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the compounds, compounds of Formula (IX) are incorporated into intracellular proteins by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein the O-RS binds to the O-tRNA Aminoacylated with a compound of Formula IX, the O-tRNA recognizes one or more selector codons of mRNA in the cell.

In certain embodiments of the compound, the cell further comprises a pylS gene and a pylT gene, and the compound of Formula IX is an intracellular protein by an aminoacyl tRNA synthetase, and a tRNA that recognizes one or more selector codons of mRNA in the cell. Incorporated herein wherein the aminoacyl tRNA synthetase is the gene product of the pylS gene and tRNA is the gene product of the pylT gene.

In certain embodiments, the selector codon is an amber codon (TAG). In certain embodiments, the cells are prokaryotic cells, while in other embodiments, the cells are eukaryotic cells. In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments, the eukaryotic cells are mammalian cells, yeast cells or insect cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cell is a CHO cell, HeLa cell or HEK293F cell. In certain embodiments, the insect cell is an sf9 cell.

Another aspect provided herein is a compound having a structure of Formula IX wherein the compound of Formula IX is 2,5-diamino-3-methylpentanoic acid or D-2,5-diamino-3-methylphene Biosynthetically produced and secreted by a growth medium comprising carbonic acid and a first cell in contact with a second cell, wherein the first cell is a feeder cell comprising a pylC gene and a pylD gene.

<Formula IX>

Figure pat00034

In certain embodiments of the compounds, 2,5-diamino-3-methylpentanoic acid is (2R, 3S) -2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the compounds, 2,5-diamino-3-methylpentanoic acid is (2R, 3R) -2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the compounds, the cell comprises a pylB gene, pylC gene and pylD gene, and the cell is ornithine, arginine, D-ornithine, D-arginine, (2S) -2-amino-6- (2 , 5-diaminopentaneamido) hexanoic acid is contacted with a growth medium comprising 2,5-diamino-3-methylpentanoic acid or D-2,5-diamino-3-methylpentanoic acid.

In certain embodiments of the compounds, the compound of Formula IX is incorporated into the protein in the second cell by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is O- The tRNA is aminoacylated with a compound of Formula IX and the O-tRNA recognizes one or more selector codons of mRNA in the second cell.

In certain embodiments of such compounds, the second cell comprises a pylS gene and a pylT gene, and the compound of Formula IX is incorporated into a protein in the second cell.

In certain embodiments of the compounds, the compound of Formula IX is incorporated into a protein in the second cell by an aminoacyl tRNA synthetase and a tRNA that recognizes one or more selector codons of mRNA in the cell, wherein the aminoacyl tRNA synthetase The gene product of the pylS gene and tRNA is the gene product of the pylT gene.

In certain embodiments, the selector codon is an amber codon (TAG).

In certain embodiments, the first cell or second cell is a prokaryotic cell. In certain embodiments, the first cell and the second cell are prokaryotic cells. In certain embodiments, the first cell or second cell is a eukaryotic cell. In certain embodiments, the first cell and the second cell are eukaryotic cells. In certain embodiments, the prokaryotic cell is an Escherichia coli cell. In certain embodiments the eukaryotic cells are mammalian cells, yeast cells or insect cells. In certain embodiments, the yeast cells are Saccharomyces cerevisiae or Pichia pastoralis cells. In certain embodiments, the mammalian cell is a CHO cell, HeLa cell or HEK293F cell. In certain embodiments, the insect cell is an sf9 cell.

Another aspect provided herein is a compound of Formula IV:

Figure pat00035

Figure pat00036

Figure pat00037

Wherein the compound having at least one polyethylene glycol (PEG) residue has an average molecular weight in the range of 1000 Da to 50 kDa, n is 20 to 1200 and exPADRE is

Figure pat00038
And PADRE is
Figure pat00039
And BG1 is
Figure pat00040
And BG2 is
Figure pat00041
Where * represents a phosphothioate linkage.

Figure 1. Structure of pyrrolysine (PYL) and pyrrolysine analogs pyrroline-carboxy-lysine (PCL: PCL-A or PCL-B).
Figure 2. Comparison of possible biosynthetic pathways for pyrrolysine (A) and PCL (B).
Aminoacylation of pylT tRNA by pyrrolysine (A) and PCL (B).
4A. A plasmid carrying pylT, pylS, pylB, pylC and pylD for incorporation of biosynthetically induced PCL or pyrrolysine in mammalian cells and a single site TAG mutant gene construct for model protein hRBP4.
4B. Single site TAG mutant gene constructs for hRBP4, mEPO, hEPO and mIgG1. Single sites are indicated by arrows.
Figure 5. The Escherichia coli (Escherichia coli ) A plasmid carrying pylT, pylS, pylB, pylC and pylD for incorporation of biosynthetically induced PCL or pyrrolysine in cells.
6A. Expression of hRBP4 in HEK293F Cells. TAG mutant construct of hRBP4 (# 1-9). Western blot by SDS-PAGE followed by anti-His antibody. Arrows at 26 kDa indicate full length hRBP4.
6B and 6C. SDS-PAGE (A) and mass spectra (B) of purified hRBP4 Phe62PCL generated in HEK293F cells in the presence of D-ornithine.
Figure 7. Mass spectrometric analysis of trypsin digest of hRBP4 Phe122PCL shows the incorporation of PCL into the target Phe122TAG site. Specified MS / MS spectrum of YWGVASF * LQK (F * = PCL) (SEQ ID NO: 17).
8. Mass spectral spectrometric analysis (TIC and EIC of 2+ ions of YWGVASF * LQK) (SEQ ID NO: 17) of the trypsin digest of hRBP4 Phe122PCL shows the incorporation of PCL into the target Phe122TAG site.
9. Mass spectrometric analysis of trypsin digests of hRBP4 Phe122PCL. Mass spectra show 3+ and 2+ precursors of YWGVASF * LQK (SEQ ID NO: 17) indicating incorporation of PCL into the target Phe122TAG site.
10. Detection of biosynthesized PCL from D-ornithine in lysate from HEK293F cells.
Incorporation of N-ε-cyclopentyloxycarbonyl-L-lysine (CYC) into various hRBP4 TAG mutant proteins in HEK293F cells. 11A shows CYC incorporation at various sites in hRBP4, as detected by SDS-PAGE, and Western blotting with anti-His-tags and anti-RBP4 antibodies. 11B shows SDS-PAGE results of purified hRBP4 Phe62CYC (mutant # 2). 11C shows the mass spectrum of hRBP4 Phe62CYC.
12. Mass spectrometric verification of CYC incorporation into the TAG site of hRBP4 mutant Phe62CYC.
13. PCL incorporation as a function of various precursors and direct incorporation of various pyrrolysine analogs (including CYC) into hRBP4 TAG mutant proteins by HEK293F cells (FIGS. 13A and 13B), and the biosynthetic genes pylB, pylC and pylD PCL incorporation using various combinations (FIG. 13C). FIG. 13A is a western blot of unpurified samples with anti-His-tagged antibodies, FIG. 13B is an SDS-PAGE gel of Ni-NTA purified protein.
14. Potential precursors for PCL biosynthesis (A) and various pyrrolysine analogs (B).
15. Evaluation of various precursor and gene combinations for incorporation of PCL into FASTE in HK100 cells.
16. Potential biosynthetic scheme for PCL (PCL-A) formation.
Figure 17A. Site specific incorporation of biosynthetically generated PCL into a single TAG coding site (4 sites) of the Fc domain of mouse IgG1.
Figure 17B. Site specific incorporation of biosynthetically generated PCL into a single TAG coding site (11 sites) of erythropoietin (EPO) as detected by SDS-PAGE.
18. Site specific incorporation of biosynthetically generated PCL into a single TAG coding site (two sites) of the thioesterase domain of human fatty acid synthetase (FAS-TE). 18 shows mass spectra (B) for SDS-PAGE (A) and PCL incorporation.
19. Site specific incorporation of biosynthetically generated PCL into a single TAG coding site (one site) of FKBP-12. 19 shows the mass spectrum (B) and determination of FKBP12-I90PCL (C) for SDS-PAGE (A) and PCL incorporation.
20. Site specific incorporation of biosynthetically generated PCL into a single TAG coding site (20 sites) of fibroblast growth factor 21 (FGF21). SDS-PAGE shows PCL incorporation into FGF21 at multiple sites.
Figure 21A shows SDS-PAGE analysis of PYL- or PCL-incorporation into mTNF-α where glutamine Gln21 (CAA) was mutated with a TAG termination codon in the presence and absence of pylB. Figure 21B SDS-PAGE to assess the purity of protein. FIG. 21C is a circular mass spectrum of mEGF Tyr10TAG expressed in Escherichia coli, indicating that PYL and PCL were incorporated into the mixture of proteins (FIG. 21C, bottom) and PCL was predominantly incorporated (FIG. 21C, top). .
22. Possible schemes for chemical derivatization of PCL with 2-amino-benzaldehyde.
23. Protein conjugates and mass changes assumed after derivatization of PCL with 2-amino benzaldehyde, 2-aminoacetophenone and 2-amino-5-nitro-benzophenone.
24. Mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-amino-benzaldehyde (2-ABA).
25. Mass spectrometric analysis of trypsin digest of 2-ABA-derivatized hRBP4 Phe122PCL protein demonstrates derivatization of PCL residues incorporated at the TAG site. YWGVASF * LQK peptide (SEQ ID NO: 17).
26. pH dependent evaluation of derivatization of hRBP4 Phe122PCL with 2-ABA.
27. Assessment of reaction efficiency as a function of the ratio of reactants to protein concentration, and reactivity of 2-ABA, 2-ANBP with 2-AAP.
28. Derivatization for (R: 4700-fold excess of 2-ABA over protein) and OMePhe incorporated hRBP4 (B: 15400-fold excess) at a molar ratio greater than 4700-fold.
29. Derivatization of FAS-TE Tyr2454PCL with 2-amino-acetophenone (2-AAP). Mass spectra of unreacted samples (A and C) and samples derivatized with 2-AAP at pH 5.0 (B) and pH 7.4 (D).
30. General scheme for site specific modification of proteins via chemical derivatization of pyrrolysine and / or PCL by 2-amino-benzaldehyde or 2-amino-benzaldehyde analogs.
31. Illustration of an embodiment of a functionalized PEG polymer, 2-amino-acetophenone-PEG8 (2-AAP-PEG8; TU3205-044) coupled to a protein via a PCL residue incorporated into the protein.
32. Derivatization of hRBP4 Phe122PCL with 2-AAP-PEG8. Unreacted hRBP4 Phe122PCL protein (C), and PCL incorporated at position 122 compared to the mass spectra of wild type hRBP4 (D and E) added with 2-AAP-PEG8 and wild type hRBP4 without 2-AAP-PEG8 added Mass spectrum at pH 7.5 (A) and ph 5.0 (B) after derivatization of hRBP4 with 2-AAP-PEG8.
33. Derivatization of FAS-TE Tyr2454PCL with 2-AAP-PEG8. Mass spectrum for unreacted protein (A), and FAS-TE Tyr2454PCL protein (B) reacted with 2-AAP-PEG8 (TU3205-044). 33C and 33D show derivatization of FAS-TE Tyr2454PCL by 2.4 kDa 2-AAP-PEG (TU3205-048) (FIG. 33C derivatizes at room temperature, FIG. 33D at 4 ° C.).
34. PEGylation of FAS-TE Tyr2454PCL with 0.5 kDa 2-AAP-PEG (2-AAP-PEG8), 2.4 kDa 2-AAP-PEG and 23 kDa 2-AAP-PEG at the indicated molar ratios.
35. Derivatization of FGF21 Lys81PCL by 2-AAP-PEG8. Mass spectrum of FGF21 Lys84PCL (B) after derivatization with unreacted FGF21 Lys84PCL (A) and 2-AAP-PEG8.
36. PEGylation of FGF21 protein. After derivatization of the seven FGF21 PCL mutants by 23 kDa 2-AAP-PEG showing PEG-FGF21, full length (FL) FGF21-PCL and truncated (TR) FGF21-PCL, before partial purification (A) and SDS-PAGE results obtained after (B).
37. PEGylation of EPO protein. SDS-PAGE after derivatization of mouse EPO PCL mutants with 23 kDa 2-AAP-PEG.
38. Derivatization of PCL with amino sugars. Generalized scheme in which a protein with PCL incorporated therein (denoted as R 1 ) is coupled with D-mannosamine.
39. Derivatization of hRBP4 Phe122PCL with D-mannosamine. Mass spectrum of hRBP4 with PCL incorporated at position 122 after reaction with mannosamine.
40. Derivatization of FAS-TE Leu2222PCL with D-mannosamine. Mass spectrum of unreacted human fatty acid synthetase (FAS-TE) (FAS-TE Leu2222PCL / Leu2223Ile) (A) and protein (B) reacted with mannosamine with PCL incorporated at position 2222.
41. Illustration of an embodiment for site specific attachment of oligosaccharides to proteins via reacting 2-ABA residues linked to oligosaccharides having PCL incorporated into the protein.
42. Illustration of certain embodiments of protein-protein conjugates (heterodimers, heterotrimers, homotrimers) formed by crosslinked proteins with PCL incorporated therein.
43. Homodimer formation by PCL specific, bifunctional crosslinker as exemplified for FGF21 Lys84PCL protein. A non-limiting example of a bifunctional crosslinker used to form a homodimer is shown in A and the mass spectrum of the reaction mixture of FGF-21 Lys84PCL crosslinked using this bifunctional linker is shown in B.
44. Homodimer formation of FGF-21 PCL mutant protein by bifunctional crosslinker. 44A shows the mass spectrum of crosslinked FGF-21, where the reaction conditions are changed from the conditions used in FIG. 43.
45. Embodiments for the crosslinking agent used to form the trimer.
46. Illustration of various embodiments of labeling using site specific labeling and the methods provided herein.
47A shows ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with biotin. 47B shows Western blot of mEGF-Y10PCL-ABA-biotin conjugates using horseradish peroxidase (HRP) conjugated goat anti-biotin antibody. 47C shows ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with fluorescein. 47D shows ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with disaccharides.
48A shows ESI mass spectrometric analysis of mTNF-Q21PCL conjugated with mono-nitrophenyl hapten. 48B shows ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with mono-nitrophenyl hapten. 48C shows ESI mass spectrometric analysis of mTNF-Q21PCL conjugated with di-nitrophenyl hapten. 48D shows ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with di-nitrophenyl hapten.
49. FIG. 49A shows ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with TLR7 agonist and FIG. 49B shows ESI mass spectrometric analysis of mEGF-Y10PCL conjugated with phospholipids.
Figures 50A and 50B show MALDI-TOF mass spectrometric analysis of mTNF-Q21PCL conjugated with PX2-PADRE at two different pH values (Figure 50A: pH 5.0; Figure 50B: pH 7.5). 50C shows ESI mass spectrometric analysis of mTNF-Q21PCL conjugated with BHA-exPADRE.
51. ESI mass spectrum showing coupling of BHA-exPADRE to mEGF-Y10PCL.
FIG. 52A is a gel transfer assay of coupling of BHA-BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Q21PCL (19.3 kDa). 52B is a gel shift assay of coupling of mHAF-Y10PCL (7.2 kDa) of BHA-BG2 (7.4 kDa).
Figure 53 illustrates an embodiment of such site specific orientation attachment.
54A shows ESI mass spectrometric analysis of hFGF21-K150PCL coupled to 2-ABA and then reduced with 20 mM NaCNBH 3 for 1 hour. 54B shows ESI mass spectrometric analysis of the reduced hFGF21-K150PCL 2-ABA conjugate after dialysis with 10 mM phosphate buffer, pH 7.5, and incubation at 50 ° C. for 1 day.
55. FIG. 55 demonstrates the stability of PCL linkages to PEGylated FGF21 in the presence and absence of reduction with NaCNBH 3 . SDS-PAGE gels for the reduced and non-reduced samples are shown in FIG. 55A. In addition, FIG. 55B shows SDS-PAGE gels for non-reduced samples incubated at 4 ° C., room temperature, 37 ° C. and 50 ° C., and 95 ° C. for 60 hours.
56. NMR analysis of PCL-A reaction with 2-ABA.
57A is a proposed structure of the product resulting from the reaction between PCL-A and 2-ABA. 57B shows the proposed equilibrium structure of the product resulting from the reaction between PCL-A and 2-ABA. 57C is a suggested structure of the reduced product.
58. NMR analysis of PCL-B reaction with 2-ABA.
59. Derivatization of pyrrolysine (Pyl) and PCL incorporated into mEGF.

Provided herein are methods and compositions for use in site-specific modification of proteins, polypeptides and / or peptides, wherein such methods are genetically encoded pyrrolysine or pyrroline-carboxy-lysine (PCL) residues, wherein , Pyrrolysine and PCL amino acids are biosynthetically produced). Provided herein are various types of molecules that are site-specifically coupled to proteins, polypeptides and / or peptides having one or more PCL residues or pyrrolysines incorporated therein biosynthetically. In certain embodiments, such site specific modifications are used to site specific label proteins, polypeptides and / or peptides. In certain embodiments, the label is a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, a chelating moiety, an insert moiety, a radioactive moiety, a chromophore moiety, a radioactive moiety, a rotating label moiety, an NMR-active moiety, a PET or MRI imaging reagent. In certain embodiments, such site specific modifications are used to attach immune modulators to proteins, polypeptides and / or peptides. In other embodiments, such site specific modifications are used to attach poly (ethylene glycol) (PEG) to proteins, polypeptides and / or peptides. In other embodiments, such site specific modifications are used to attach (glycosylate) sugars to proteins, polypeptides and / or peptides.

In other embodiments, such site specific modifications are used to crosslink proteins, polypeptides and / or peptides, thereby forming heterooligomers including but not limited to heterodimers and heterotrimers. In certain embodiments, such site specific modifications are used to site specific crosslink the antibody to a protein, polypeptide and / or peptide. In other embodiments, such site-specific modifications crosslink proteins, polypeptides, and / or peptides such that protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates, polypeptide-peptide conjugates or peptides -Used to form peptide conjugates.

In other embodiments, such site specific modifications are used to site-specifically link an antibody to a toxic protein provided herein, thereby forming an antibody-drug conjugate. In other embodiments, such site-specific modifications are used to site-specifically link an antibody coupled to a low molecular weight drug to a protein, thereby forming an antibody-drug conjugate.

In other embodiments, such site specific modifications are used to site-specifically link receptor-ligand to toxic proteins provided herein, thereby forming receptor-ligand-drug conjugates. In other embodiments, such site-specific modifications are used to site-specifically link receptor-ligands coupled to low molecular weight drugs to proteins, thereby forming receptor-ligand-drug conjugates.

Provided herein are proteins, polypeptides and / or peptides having pyrrolysine and / or PCL incorporated therein using the provided methods. These proteins include erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6) , Interleukin 10 (IL-10), interleukin 17 (IL-17), insulin-like growth factor 1 (IGF-1), and interferon beta (INF-β).

Provided herein are proteins, polypeptides and / or peptides having pyrrolysine and / or PCL incorporated therein using the methods provided herein and further derivatized using the methods provided herein. Such derivatizations include, but are not limited to, PEGylation. These proteins include erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6) , Interleukin 10 (IL-10), interleukin 17 (IL-17), insulin-like growth factor 1 (IGF-1), and interferon beta (INF-β).

Further provided herein are proteins, polypeptides and / or peptides crosslinked using the methods provided herein having pyrrolysine and / or PCL incorporated therein. Such proteins include, but are not limited to, erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α) and interferon beta (INF-β).

In other embodiments, such site-specific modifications are proteins in which the location of site-specific incorporated pyrrolysine or PCL allows for controlled orientation and attachment of the protein, polypeptide and / or peptide onto the solid support surface. , To produce polypeptides and / or peptides. In certain embodiments, such solid supports are coated or uncoated plastic microtiter plates, glass slides, silica surfaces, polymer beads, gold particles or nano-particles. In certain embodiments, such controlled orientation and attachment is used for analysis of proteins, polypeptides and peptides by ELISA or other antibody assays. In certain embodiments, such controlled orientation and attachment is used to purify and / or identify ligands of immobilized proteins, polypeptides and / or peptides. In certain embodiments, such controlled orientation and attachment is achieved by protein, polypeptide and peptide, by evanescent wave analysis, including, but not limited to, assays that do not use labels using surface plasmon resonance analysis, And their interactions. In certain embodiments, such controlled orientation and attachment on a surface can include microbalances (electrical, optical and / or mechanical), infrared spectroscopy, Raman spectroscopy (surface enhanced Raman spectroscopy, disappearance resonance, fluorescence, interferometry , Mass spectrometry and other spectroscopy) are used to analyze proteins, polypeptides and peptides, and their interactions. Such analytical methods may be useful for immobilizing proteins, polypeptides and / or peptides, other proteins, polypeptides, peptides, nucleic acids, DNA, RNA, small molecules, drugs, metabolites, sugars, carbohydrates, oligosaccharides, polysaccharides and / or other It is used to investigate the interactions with molecules, including the change in coordination caused by such interactions. This method of assay also examines the interaction of immobilized proteins, polypeptides and / or peptides with multiple sub-unit protein complexes, studies natural, recombinant, synthetic or tagged recombinant molecules, body fluids, cell cultures. Discover new interaction partners in supernatants or raw extracts of water, study the interaction of small molecules such as drug candidates with their targets, and use membranes, artificial membranes or vesicles to membrane biochemical or membrane-bound receptor interactions Study the action, investigate replication, transcription and translation, determine molecular relationships during the formation of protein complexes and their interaction with DNA, study hybridization of DNA and RNA, and interact with whole cells or viruses , The effects of glycosylation on molecular interactions, and the specific recognition characteristics of cell surface carbohydrates It is used to determine.

In other embodiments, such site specific modifications are used to site specificly attach nucleic acids to proteins. In certain embodiments, such site specific modifications are used to site specificly attach a nucleic acid to an antibody or antibody fragment. In certain embodiments, the nucleic acid attached to the protein or antibody is used to immobilize the protein or antibody at a defined position on the DNA array through hybridization. In certain embodiments, the nucleic acid attached to the protein or antibody is PCR, strand displacement amplification (SDA), ligase chain reaction (LCR), contiguous ligation immuno-PCR, rolling circle amplification, transcription mediated amplification, tyramide signal of NEN Used to detect protein or antibody binding through amplification or other signal amplification methods. In certain embodiments, such site specific attachment of an antibody to a PCR probe is used to generate reagents for an immune-PCR reaction (M. Adler, R. Wacker, Ch. M. Niemeyer, Sensitivity by combination : Immuno-PCR and related technologies, Analyst, 2008, 133, 702-718). In certain embodiments, nucleic acids attached to proteins or antibodies allow for simultaneous immuno-PCR or other immuno-assay (multiplexed immune-PCR or multiplexed immune-assay) of multiple analytes.

In certain embodiments, the nucleic acid attached to a protein or antibody modulates the formation of homo- and heterodimers.

Justice

The term "alkyl" as used herein refers to saturated branched or straight chain hydrocarbons. As used herein, the terms “C 1 -C 3 alkyl”, “C 1 -C 4 alkyl”, “C 1 -C 5 alkyl”, “C 1 -C 6 alkyl”, “C 1 -C 7 alkyl” and "C 1 -C 8 alkyl" refers to an alkyl group containing at least one, and up to 3, 4, 5, 6, 7, or 8 carbon atoms, respectively. Non-limiting examples of alkyl groups used herein include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, hexyl, heptyl, octyl, Nonyl, decyl and the like.

As used herein, the term "alkylene" refers to a saturated branched or straight chain divalent hydrocarbon radical, where the radical is derived by removing one hydrogen atom from each two carbon atoms. As used herein, the terms “C 1 -C 3 alkylene”, “C 1 -C 4 alkylene”, “C 1 -C 5 alkylene” and “C 1 -C 6 alkylene” are one or more, and It refers to alkylene groups each containing up to 3, 4, 5 or 6 carbon atoms. Non-limiting examples of alkylene groups used herein include methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, t-butylene, n-pentylene, isopentylene , Hexylene and the like.

As used herein, the term "alkoxy" refers to the group -OR a , where R a is an alkyl group as defined herein. As used herein, the terms “C 1 -C 3 alkoxy”, “C 1 -C 4 alkoxy”, “C 1 -C 5 alkoxy”, “C 1 -C 6 alkoxy”, “C 1 -C 7 alkoxy” and "C 1 -C 8 alkoxy" refers to an alkoxy group having at least one alkyl moiety and no more than 3, 4, 5, 6, 7 or 8 carbon atoms each. Non-limiting examples of alkoxy groups as used herein include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, pentoxy, hexoxy, heptoxy and the like.

As used herein, the term “amino terminal modifying group” refers to any molecule that forms a link with a terminal amine group. By way of example, such terminal amine groups include, but are not limited to, amine protecting groups, the ends of polymer molecules, where such polymer molecules include polypeptides, polynucleotides, polysaccharides, and the like. Amino terminal modifying groups also include, but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminal modifying groups include polyethylene glycol or serum albumin. Certain amino terminal modifying groups are used to modify the therapeutic properties of a protein, including but not limited to increasing serum half-life.

As used herein, the term “aryl” is a monocyclic, bicyclic, having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic, and each ring in the system contains 3 to 7 ring members And tricyclic ring systems. Aryl groups are "optionally substituted" where such aryl groups contain one or more substituents. Unless defined otherwise herein, suitable substituents are generally halogen, -R, -OR, -SR, -NO 2 , -CN, -N (R) 2 , -NRC (O) R, -NRC (S) R, -NRC (O) N (R) 2 , -NRC (S) N (R) 2 , -NRCO 2 R, -NRNRC (O) R, -NRNRC (O) N (R) 2 , -NRNRCO 2 R, -C (O) C (O) R, -C (O) CH 2 C (O) R, -CO 2 R, -C (O) R, -C (S) R, -C (O) N (R) 2 , -C (S) N (R) 2 , -OC (O) N (R) 2 , -OC (O) R, -C (O) N (OR) R, -C (NOR ) R, -S (O) 2 R, -S (O) 3 R, -SO 2 N (R) 2 , -S (O) R, -NRSO 2 N (R) 2 , -NRSO 2 R,- N (OR) R, -C (= NH) -N (R) 2 , -P (O) 2 R, -PO (R) 2 , -OPO (R) 2 ,-(CH 2 ) 0-2 NHC (O) R, phenyl optionally substituted with R (Ph), -O (Ph) optionally substituted with R,-(CH 2 ) 1-2 (Ph) optionally substituted with R, or-optionally substituted with R CH = CH (Ph), wherein each occurrence of R is hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkoxy, unsubstituted 5 to 6 membered heteroaryl, Phenyl, -O (Ph) or -CH 2 (Ph), or on the same or different substituents Two independently appearing R's, combined with the atom (s) to which each R is bonded, are optionally substituted 3-12 membered saturated, partially unsaturated having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur Or form a fully unsaturated monocyclic or bicyclic ring. Non-limiting examples of aryl groups as used herein include phenyl, naphthyl, fluorenyl, indenyl, azulenyl, anthracenyl and the like.

The term "arylene" as used means a divalent radical derived from an aryl group.

As used herein, "bifunctional linker" (also referred to as "bifunctional polymer") refers to a linker comprising two functional groups that can specifically react with other moieties to form covalent or non-covalent bonds. Such residues include, but are not limited to, amino acid side chain groups. By way of example only, a bifunctional linker has a functional group reactive with a group on the first peptide, and another functional group reactive with a group on the second peptide, and thus a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. To form. Bifunctional linkers have any desired length or molecular weight and are selected to provide the particular spacing or configuration desired.

As used herein, "multifunctional linker" (also referred to as "multifunctional polymer") refers to a linker comprising two or more functional groups that can react with other moieties to form covalent or non-covalent bonds. Such residues include, but are not limited to, amino acid side chain groups. The multifunctional linker has any desired length or molecular weight and is chosen to provide the particular spacing or configuration desired.

As used herein, the term "cyano" refers to the group -CN.

As used herein, the term “cycloalkyl” refers to a saturated or partially unsaturated, monocyclic, fused bicyclic, fused tricyclic or crosslinked polycyclic ring assembly. As used herein, the terms "C 3 -C 5 cycloalkyl", "C 3 -C 6 cycloalkyl", "C 3 -C 7 cycloalkyl", "C 3 -C 8 cycloalkyl", "C 3 -C 9 cycloalkyl "and" C 3 -C 10 cycloalkyl "include at least 3 saturated or partially unsaturated, monocyclic, fused bicyclic or crosslinked polycyclic ring assemblies, and 5, 6, 7, 8, It refers to a cycloalkyl group containing up to 9 or 10 carbon atoms. Non-limiting examples of cycloalkyl groups as used herein include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, decahydronaphthalenyl, 2,3,4,5,6,7-hexahydro-1H-indenyl and the like.

As used herein, the term “cyclodextrin” refers to a cyclic carbohydrate consisting of 6 to 8 or more glucose molecules in ring formation. The outside of the ring contains a water soluble group, and at the center of the ring there is a relatively nonpolar cavity that can accept small molecules.

The term "halogen" as used herein refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

As used herein, the term "halo" refers to halogen radicals (fluoro (-F), chloro (-Cl), bromo (-Br) and iodo (-I)).

The term "haloacyl" as used herein includes halogen residues, including but not limited to, -C (O) CH 3 , -C (O) CF 3 , -C (O) CH 2 OCH 3 , and the like. It refers to an acyl group.

As used herein, the term “haloalkyl” or “halo-substituted alkyl” refers to an alkyl group, as defined herein, substituted with one or more halo groups or combinations thereof. Non-limiting examples of such branched or straight chain haloalkyl groups as used herein include, but are not limited to, trifluoromethyl, pentafluoroethyl, and the like, substituted with one or more halo groups or combinations thereof. Methyl, ethyl, propyl, isopropyl, isobutyl and n-butyl.

As used herein, the term “haloalkoxy” refers to an alkoxy group, as defined herein, substituted with one or more halo groups or combinations thereof. Non-limiting examples of such branched or straight chain haloalkoxy groups as used herein include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, substituted with one or more groups or combinations thereof. t-butoxy, pentoxy, hexoxy, heptoxy and the like.

As used herein, the term “heteroalkyl” refers to an alkyl group, as defined herein, wherein one or more carbon atoms are independently replaced by one or more oxygen, sulfur, nitrogen, or combinations thereof.

The term "heteroalkylene" as used herein refers to a divalent radical derived from heteroalkyl.

The term "heteroaryl" as used herein has a total of 5 to 14 ring members, at least one ring in the system is aromatic, and at least one ring in the system contains at least one heteroatom selected from nitrogen, oxygen and sulfur. And monocyclic, bicyclic and tricyclic ring systems where each ring in the system contains 3 to 7 ring members. Unless otherwise defined above and herein, suitable substituents on unsaturated carbon atoms of heteroaryl groups are generally halogen, -R, -OR, -SR, -NO 2 , -CN, -N (R) 2 , -NRC (O) R, -NRC (S) R, -NRC (O) N (R) 2 , -NRC (S) N (R) 2 , -NRCO 2 R, -NRNRC (O) R, -NRNRC (O ) N (R) 2 , -NRNRCO 2 R, -C (O) C (O) R, -C (O) CH 2 C (O) R, -CO 2 R, -C (O) R, -C (S) R, -C (O) N (R) 2 , -C (S) N (R) 2 , -OC (O) N (R) 2 , -OC (O) R, -C (O) N (OR) R, -C (NOR) R, -S (O) 2 R, -S (O) 3 R, -SO 2 N (R) 2 , -S (O) R, -NRSO 2 N ( R) 2 , -NRSO 2 R, -N (OR) R, -C (= NH) -N (R) 2 , -P (O) 2 R, -PO (R) 2 , -OPO (R) 2 ,-(CH 2 ) 0-2 NHC (O) R, phenyl (Ph) optionally substituted with R, -O (Ph) optionally substituted with R,-(CH 2 ) 1-2 (optionally substituted with R) Ph), or —CH═CH (Ph) optionally substituted with R, wherein each occurrence of R independently is hydrogen, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkoxy, selected from unsubstituted 5 to 6 membered heteroaryl, phenyl, -O (Ph), or -CH 2 (Ph) being Or, two Rs, which appear independently on the same substituent or on different substituents, are combined with the atom (s) to which each R is bonded, optionally substituted with 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur To form 3- to 12-membered saturated, partially unsaturated or fully unsaturated monocyclic or bicyclic rings. Non-limiting examples of heteroaryl groups as used herein include benzofuranyl, benzofurazanyl, benzoxazolyl, benzopyranyl, benzthiazolyl, benzothienyl, benzazinyl, benzimidazolyl, benzothiopyra Nyl, Benzo [1,3] dioxol, Benzo [b] furyl, Benzo [b] thienyl, cinnaolinyl, furazinyl, furyl, furopyridinyl, imidazolyl, indolyl, indolinyl, indolin 2-one, indazolyl, isoindolinyl, isoquinolinyl, isoxazolyl, isothiazolyl, 1,8-naphthyridinyl, oxazolyl, oxindadolyl, oxadizolyl, pyrazolyl, pyrrolyl, Phthalazinyl, pterridinyl, purinyl, pyridyl, pyridazinyl, pyrazinyl, pyrimidyl, pyrimidinyl, quinoxalinyl, quinolinyl, quinazolinyl, 4H-quinolininyl, thiazolyl, thia Diazolyl, thienyl, triazinyl, triazolyl and tetrazolyl.

As used herein, the term “heterocycloalkyl” means that at least one of the ring carbons is —O—, —N═, —NR—, —C (O) —, —S—, —S (O) — or —S (O) 2 -wherein R is a hydrogen, a C 1 -C 4 alkyl or nitrogen protecting group substituted by a moiety provided that the ring of the group does not contain two adjacent O or S atoms It refers to cycloalkyl as defined in. Non-limiting examples of heterocycloalkyl groups as used herein include morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, 1,4-dioxa-8- Aza-spiro [4.5] des-8-yl, 2H-pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, 1,3-dioxolanyl, 2-imidazolinyl, imidazolidinyl, 2 -Pyrazolinyl, pyrazolidinyl, 1,4-dioxanyl, 1,4-ditianyl, thiomorpholinyl, azepanyl, hexahydro-1,4-diazepynyl, tetrahydrofuranyl, dihydro Furanyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, thioxanyl, azetidinyl, oxetanyl, thietanyl, oxepanyl, thiepanyl, 1,2, 3,6-tetrahydropyridinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, dithianil, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydro Furanyl, imidazolinyl, imidazolidinyl, 3-azabicyclo [3.1.0] hexanyl and 3-azabicyclo [4.1.0] heptanyl.

As used herein, the term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon.

The term "hydroxyl" as used herein refers to an -OH group.

As used herein, the term “hydroxyalkyl” refers to an alkyl group, as defined herein, substituted with one or more hydroxyls, where hydroxyl is as defined herein. Non-limiting examples of branched or straight chain “C 1 -C 6 hydroxyalkyl groups include methyl, ethyl, propyl, isopropyl, isobutyl, and n-butyl substituted independently with one or more hydroxyl groups. .

As used herein, the term “optionally substituted” means that the groups mentioned are alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl From amino, including thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, and mono- and disubstituted amino groups, and protected derivatives thereof It may or may not be substituted with one or more additional group (s) selected individually and independently. Non-limiting examples of optional substituents include halo, -CN, -OR, -C (O) R, -OC (O) R, -C (O) OR, -OC (O) NHR, -C (O) N (R) 2 , -SR-, -S (= O) R, -S (= O) 2 R, -NHR, -N (R) 2 , -NHC (O)-, NHC (O) O- , -C (O) NH-, S (= O) 2 NHR, -S (O) 2 N (R) 2 , -NHS (= O) 2 , -NHS (O) 2 R, C 1 -C 6 Alkyl, C 1 -C 6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halo-substituted C 1 -C 6 alkyl, halo-substituted C 1 -C 6 alkoxy, where each R is H , Halo, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, halo-substituted C 1 -C 6 alkyl, halo-substituted C 1 -C 6 alkoxy Independently selected from).

As used herein, the term "affinity label" refers to a label that binds to another molecule reversibly or irreversibly.

As used herein, the term "amber codon" refers to the incorporation site of pyrrolysine, PCL and other pyrrolysine analogs and corresponds to UAG, which is a nucleotide triplet in messenger RNA. The nucleotide sequence TAG is encoded in DNA and transcribed into UAG in RNA translated into protein. TAG and UAG codons are used interchangeably herein to refer to the site of incorporation of pyrrolysine, PCL and other pyrrolysine analogs.

As used herein, the term “amino acid” refers to naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimetics that function in a similar manner to naturally occurring amino acids, provided that their structures allow D and L stereoisomeric forms. In all stereoisomeric forms). Amino acids are referred to herein by their names, their commonly known three letter symbols, or the one letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as encoded amino acids that are subsequently modified. Naturally occurring amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr), Valine (Val), pyrrolysine (Pyl), selenocysteine (Sec) and pyrroline-carboxy-lysine (PCL), including but not limited to. Modified encoded amino acids include hydroxyproline, γ-carboxyglutamate, O-phosphoseline, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-amino Butyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminobutyric acid, 3-aminobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, Desmocin, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3 Hydroxyproline, 4-hydroxyproline, isodesmocin, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, nord Valine, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline; It is not. The term amino acid also includes naturally occurring amino acids in certain organisms that are metabolites but not encoded by genetic code for incorporation into proteins. Such amino acids include, but are not limited to, ornithine, D-ornithine and D-arginine.

As used herein, the term “amino acid analog” refers to a compound having a basic chemical structure, such as a naturally occurring amino acid, by way of example only having an α-carbon bound to hydrogen, carboxyl groups, amino groups and R groups. Amino acid analogs include natural and non-natural amino acids that are chemically blocked reversibly or irreversibly, or chemically modified at their C-terminal carboxyl groups, their N-terminal amino groups and / or their side chain functional groups. Such analogues include methionine sulfoxide, methionine sulfone, S- (carboxymethyl) -cysteine, S- (carboxymethyl) -cysteine sulfoxide, S- (carboxymethyl) -cysteine sulfone, aspartic acid- (beta-methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and methionine methyl sulfonium. Specific blocking agents include, but are not limited to, t-butyloxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc).

As used herein, the term "amino acid mimetic" refers to a compound that has a structure different from the general chemical structure of an amino acid, but which functions in a similar manner to naturally occurring amino acids.

As used herein, the term “non-natural amino acid” is intended to denote an amino acid structure that cannot be biosynthetically produced in any organism (whether identical or different) using an unmodified or modified gene from any organism. do. In addition, it is understood that such "non-natural amino acids" require modified tRNA and modified tRNA synthetase (RS) for incorporation into proteins. Such "selected" orthogonal tRNA / RS pairs are specific to non-natural amino acids and are generated by a selection procedure or similar procedure developed by Schultz et al. By way of example, pyrroline-carboxy-lysine is a "natural amino acid" in that it is biosynthesically produced by a gene transferred from one organism to a host cell and in that it is incorporated into the protein using native tRNA and tRNA synthetase genes. P-aminophenylalanine (Generation of a bacterium with a 21 amino acid genetic code, Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG. J Am Chem Soc. 2003 Jan 29; 125 (4): 935-9], although biosynthetically produced, are “non-natural amino acids” because they are incorporated into proteins by “selected” orthogonal tRNA / tRNA synthetase pairs.

As used herein, the term "amino acid residue"

Figure pat00042
Refers to a moiety having the structure of wherein the moiety is derived from an amino acid and the R group is the side chain of any amino acid described herein. Such amino acid residues include alaninyl, argininyl, asparaginyl, aspartyl, cysteinyl, glutamyl, glutamyl, glycinyl, histidinyl, isoleucinyl, leucineyl, ricinyl, Methioninyl, phenylalaninyl, prolinyl, serinyl, threonyl, tryptophanyl, tyrosinyl, valenyl, pyroglutamate, formylmethionine, pyroglycinyl and selenocysteinyl However, the present invention is not limited thereto.

The term "amino terminal modification group" refers to any molecule that can be attached to a terminal amino group. Such amino terminal modification groups include, but are not limited to, amine protecting groups at the ends of the polymer molecule, and include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modifying groups also include, but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, amino terminal modification groups include polyethylene glycol or serum albumin. Certain amino terminal modification groups are used to modify the therapeutic properties of a protein, polypeptide or peptide, for example, but to increase the serum half-life of such protein, polypeptide or peptide.

As used herein, the term “antibody fragment” refers to any form of antibody other than full length. Antibody fragments herein include antibodies that are smaller components present in full length antibodies, and engineered antibodies. Antibody fragments include Fv, Fc, Fab and (Fab ') 2 , single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, frames Work regions, constant regions, heavy chains, light chains, and variable regions, and alternative scaffold non-antibody molecules, bispecific antibodies, and the like. Another functional substructure is the single chain Fv (scFv) consisting of the variable regions of immunoglobulin heavy and light chains covalently bound by a peptide linker. Such small (MW <25,000 Da) proteins generally maintain specificity and affinity for the antigen in a single polypeptide and can provide a convenient building block for large antigen-specific molecules. Unless specifically stated otherwise, the content and claims using the term “antibody” or “antibodies” also include “antibody fragments” and “antibody fragments”.

As used herein, the term “bioavailability” refers to the proportion and extent to which a substance or active moiety thereof is delivered from a pharmaceutical dosage form and available at the site of action or in general circulation. An increase in bioavailability refers to an increase in the rate and extent by which a substance or active moiety thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in general circulation. For example, an increase in bioavailability may be indicated by an increase in the concentration of a substance or active moiety in the blood relative to other substances or active moieties.

As used herein, the term “biologically active molecule”, “biologically active moiety” or “biologically active agent” refers to an organism such as, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. It refers to any substance that can affect any physical or biochemical property of a biological system, pathway, molecule or interaction in connection with. In particular, biologically active molecules as used herein include any substance intended for the diagnosis, healing, alleviation, treatment or prevention of a disease in humans or other animals, or otherwise for the improvement of the physical or mental well being of a human or animal. However, it is not limited thereto. Examples of biologically active molecules include peptides, proteins, enzymes, DNA, RNA, small molecule drugs, hard drugs, soft drugs, polysaccharides, oligosaccharides, disaccharides, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides Seeds, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of biologically active agents suitable for use in the methods and compositions described herein include drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, antitumor agents, cardiovascular Agonists, anti-anxiety agents, hormones, growth factors, steroidal agents, microvials-induced toxins, and the like.

The term "modulation of biological activity" as used herein increases or decreases the concentration or reactivity of a protein, polypeptide, peptide, DNA, RNA, saccharide, sugar, metabolite, precursor, cofactor or other biologically active chemical or entity. Altering the selectivity of a protein, polypeptide, peptide, DNA, RNA, saccharide, sugar, metabolite, precursor, cofactor or other biologically active chemical or entity, or altering the protein, polypeptide, peptide, DNA, RNA , Refers to improving or reducing substrate selectivity of saccharides, sugars, metabolites, precursors, cofactors or other biologically active chemicals or entities.

As used herein, the term “biomaterial” refers to a material derived from a biological, such as, but not limited to, a material obtained from a bioreactor and / or from recombinant methods and techniques.

The term "biophysical probe" as used herein refers to a probe that detects or monitors structural changes in a molecule by physical detection methods. Such molecules include, but are not limited to, proteins, polypeptides, peptides, DNA or RNA. Such “biophysical probes” are also used to detect or monitor the interaction of proteins, polypeptides, peptides, DNA or RNA with other molecules such as, but not limited to, macromolecules. Examples of biophysical probes include, but are not limited to, molecular mass, nuclear spin, UV absorbance, fluorescence, circular dichroism, heat capacity, melting temperature or other endogenous molecular properties. Examples of biophysical probes also include labels added to the molecule. Such probes include, but are not limited to, spin labels, fluorophores, isotope labels and photoactive groups.

As used herein, the term “biosynthetically produced” refers to any method that uses cells or enzymes to produce amino acids. Such methods include the use of at least one of a precursor and an enzyme. In certain embodiments, such amino acids are incorporated into the protein. In certain embodiments biosynthesis and incorporation of amino acids occurs in the same cell, while in other embodiments amino acids are biosynthetically produced in separate cells (“feeder cells”) or in separate cell cultures so that the amino acid is another cell. Is incorporated into the protein. In the latter case, the amino acids are optionally purified from separate cell cultures, then the purified amino acids are added to the medium of the cell culture incorporating the amino acids into the protein.

As used herein, the term “biotin analogue” (also referred to as a “biotin mimetic”) refers to any molecule other than biotin that binds with high affinity with avidin and / or streptavidin.

The term "carboxy terminal modifying group" refers to any molecule that can be attached to a terminal carboxy group. Such carboxy end groups include, but are not limited to, carboxylate protecting groups at the ends of polymer molecules, and such polymer molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminal modifying groups also include, but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminal modifying groups include polyethylene glycol or serum albumin. Certain carboxy terminus modifying groups are used to modify the therapeutic properties of a protein, polypeptide or peptide, eg, to increase serum half-life.

As used herein, the term “chemically cleavable group” (also referred to as “chemically labile”) refers to a group that breaks or cleaves upon exposure to acids, bases, oxidants, reducing agents, chemical initiators, or radical initiators.

As used herein, the term “chemiluminescent group” refers to a group that emits light as a result of a chemical reaction without applying heat. By way of example only, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is reacted with an oxidizing agent such as hydrogen peroxide (H 2 O 2 ) in the presence of a base and a metal catalyst State product (3-aminophthalate, 3-APA) is produced and subsequently detectable light is emitted.

As used herein, the term “chromophore” refers to a molecule that absorbs light of visible, UV or IR wavelengths.

As used herein, the term "cofactor" refers to an atom or molecule essential for the action of a large molecule. Cofactors include, but are not limited to, inorganic ions, coenzymes, proteins, or some other factor necessary for the activity of the enzyme.

As used herein, the term “cofolding” refers to a refolding process, reaction, or method that uses two or more molecules that interact with each other to transform an unfolded or improperly folded molecule into an appropriately folded molecule. By way of example only, “co-folding” uses two or more polypeptides that interact with each other to transform an unfolded or improperly folded polypeptide into the original properly folded polypeptide.

As used herein, the term "cytotoxic" refers to a compound that harms a cell.

As used herein, the term “denaturing agent” or “denaturing agent” refers to any compound or substance that results in reversible unfolding of a protein. The strength of the denaturing agent or denaturing agent will be determined by both the nature and concentration of the particular denaturing agent or denaturing agent. By way of example, denaturing agents or denaturants include, but are not limited to, chaotropes, detergents, organic, water miscible solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine and sodium thiocyanate. Non-limiting examples of detergents include rigid detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (eg, twin or triton cleaners), sarcosyls, mild non-ionic detergents (eg, digitonin), Mild cationic detergents such as N-2,3- (dioleyloxy) -propyl-N, N, N-trimethylammonium, mild ionic detergents (eg sodium cholate or sodium deoxycholate) or amphoteric detergents Such as, but not limited to, sulfobetaine (Zwittergent), 3- (3-chloramidopropyl) dimethylammonio-1-propane sulfate (CHAPS) and 3- (3-chloramido Propyl) dimethylammonio-2-hydroxy-1-propane sulfonate (CHAPSO), including but not limited to. Non-limiting examples of organic, water miscible solvents include, but are not limited to, acetonitrile, lower alkanols (especially C 2 -C 4 alkanols such as ethanol or isopropanol) or lower alkanediols (C 2 -C 4 alkanediols such as ethylene-glycol) can be used as the modifier. Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or dipetanoylphosphatidylcholine.

As used herein, the term “detectable label” refers to analytical techniques such as, but not limited to fluorescence, chemiluminescence, electron-rotation resonance, ultraviolet / visible absorbance spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic Refers to labels that can be observed using resonance, radiometric methods, and electrochemical methods.

As used herein, the term “drug” refers to any substance used to prevent, diagnose, alleviate, treat or cure a disease or condition.

As used herein, the term "dye" refers to a soluble colored material containing chromophores.

As used herein, the term “electron high density group” refers to a group that scatters electrons when irradiated with an electron beam. These groups include ammonium molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, anhydrous indium trichloride, lanthanum nitrate, and lead acetate trihydrate. , Lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver protein compound (Ag assay: 8.0-8.5%) ", Silver tetraphenylphosphine (S-TPPS), sodium chloroacetate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate and vanadil sulfate It is not limited to this.

As used herein, the term “energy transfer agent” refers to a molecule capable of donating or accepting energy from another molecule. By way of example only, fluorescence resonance energy transfer (FRET) is characterized in that the excited state energy of a fluorescent donor molecule is transferred to a receiving molecule that is not excited by non-radioactivity, and then emits the donated energy by fluorescence at longer wavelengths. Is a dipole-dipole coupling process.

The term "improve" or "improve" means increasing and also prolonging the desired effect in terms of efficacy or duration. By way of example, "improving" the effect of a therapeutic agent refers to the ability to increase or prolong the effect of the therapeutic agent in terms of efficacy or duration during treatment of the disease, disorder or condition. As used herein, “improved effective amount” refers to an amount suitable for improving the effectiveness of a therapeutic agent in the treatment of a disease, disorder or condition. When used in a patient, the amount effective for such use will depend on the severity and course of the disease, disorder or condition, existing therapy, the patient's health condition and response to the drug, and the judgment of the treating physician.

The term “eukaryotic” refers to organisms belonging to the phylogenetic region Eucarya, such as but not limited to animals (such as, but not limited to mammals, insects, reptiles and birds), ciliforms, plants (Such as, but not limited to, monocotyledonous plants, dicotyledons and algae), fungi, yeasts, flagellas, microspores, and protozoa.

As used herein, the term “fatty acid” refers to a carboxylic acid having a hydrocarbon side chain of at least about C 6 .

As used herein, the term “fluorophore” refers to a molecule that emits photons upon excitation and is therefore fluorescent.

The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive moiety" are somewhat synonymous in the chemical industry and perform some function or activity. And a portion of a molecule that is reactive with other molecules.

The term "identity" as used herein refers to two or more sequences or subsequences that are identical. Moreover, as used herein, the term “substantially identical” refers to the same sequence unit when compared and aligned for maximum correspondence over a comparison range or designated area, as measured using a comparison algorithm or by manual alignment and visual inspection. Refers to two or more sequences having a percentage. By way of example only, two or more sequences indicate that the sequence unit is about 60% identity, about 65% identity, about 70% identity, about 75% identity, about 80% identity, about 85% identity, about 90 over a specific site. It may be "substantially the same" if it is% identity or about 95% identity. This percentage describes the "% identity" of two or more sequences. Identity of a sequence may exist over a region that is at least about 75-100 sequence units in length, over a region that is about 50 sequence units in length, or throughout the entire sequence unless otherwise specified. This definition also refers to the complement of the test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are identical, whereas two or more polypeptide sequences are about 60% identity, about 65% identity, about 70% identity over a specific site. , Substantially 75% identity, about 80% identity, about 85% identity, about 90% identity or about 95% identity. Identity can exist over a region that is at least about 75-100 amino acids in length, over a region that is about 50 amino acids in length, or throughout the entire sequence of a polypeptide sequence unless otherwise specified. Moreover, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are identical, whereas two or more polynucleotide sequences are about 60% identical, about 65% identical, “Substantially the same” when about 70% identity, about 75% identity, about 80% identity, about 85% identity, about 90% identity or about 95% identity. Identity can exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or throughout the entire sequence of the polynucleotide sequence unless otherwise specified.

The term “immunogenic” as used herein refers to an antibody response to the administration of a therapeutic drug. Immunogenicity for therapeutic proteins, polypeptides and peptides provided herein is obtained using quantitative and qualitative assays for detecting antibodies to the therapeutic proteins, polypeptides and peptides in biological fluids. Such assays include, but are not limited to, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), luminescent immunoassay (LIA), and fluorescence immunoassay (FIA). This analysis of immunogenicity compares the antibody response upon administration of the therapeutic proteins, polypeptides and peptides provided herein to the antibody response upon administration of a control therapeutic protein, polypeptide or peptide or upon administration of a delivery vehicle or delivery buffer. It includes.

As used herein, the term “inserting agent” (also referred to as an “insertable group”) refers to a molecule or group that is inserted in an intermolecular space between another molecule or between molecules. By way of example only, an insert or insert group may be a molecule inserted into the stacked base of a DNA double helix.

As used herein, the term "label" refers to a substance that is incorporated into a compound and is easily detected, so that its physical distribution can be detected and / or monitored.

As used herein, the term “linking portion” refers to a bond or chemical moiety formed from a chemical reaction between a functional group of a first molecule and a functional group of a second molecule. Such linkages include, but are not limited to, covalent and non-covalent linkages, and such chemical moieties include esters, carbonates, imine phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, oligonucleotide linkages and Table 1 herein. But are not limited to those set forth in. "Hydrolytically stable linkage" means that the linkage is substantially stable in water and does not react with water under physiological conditions for extended periods of time at, for example, but not limited to, the pH value used. "Hydrolytically unstable or degradable linkage" means that the linkage is degradable in water or in an aqueous solution, such as blood. "Enzymatically unstable or degradable linkage" means that the linkage is degraded by one or more enzymes. By way of example only, certain PEGs and related polymers include linkages that are degradable in the polymer backbone or in the linking group between the PEG polymer backbone and one or more terminal functional groups of the proteins, polypeptides or peptides provided herein. Such degradable linkages include, but are not limited to, ester linkages formed by reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on the biologically active agent, and such ester groups are generally hydrolyzed under physiological conditions to Release. Other hydrolytically degradable linkages include carbonate linkages; Imine linkages resulting from the reaction of amines with aldehydes; Phosphate ester linkages formed by the reaction of an alcohol with a phosphate group; Hydrazone linkages which are reaction products of hydrazide and aldehydes; An acetal connecting part which is a reaction product of aldehyde and alcohol; An orthoester connecting part which is a reaction product of formate and alcohol; Peptide linkages formed by amine groups, including but not limited to polymers such as amine groups at the ends of PEG, and carboxyl groups of peptides; And oligonucleotide linkages formed by, but not limited to, phosphoramidite groups, including but not limited to, phosphoramidite groups at the ends of the polymer, and 5 'hydroxyl groups of oligonucleotides. Do not.

The term "medium" or "medium" as used herein refers to any culture medium used to grow and harvest cells and / or products expressed and / or secreted by such cells. Such “medium” or “medium” includes any host cell, only by way of example bacterial host cell, yeast host cell, insect host cell, plant host cell, eukaryotic host cell, mammalian host cell, CHO cell, prokaryotic host. Cells, Escherichia coli, or Pseudomonas host cells, and solutions, solid, semisolid, or rigid supports that may supplement or contain the cell contents. Such “medium” or “medium” includes, but is not limited to, a medium or medium in which host cells have been grown to secrete polypeptides, including but not limited to, medium before or after the proliferation step. Such “medium” or “mediums” also include, but are not limited to, buffers or reagents containing host cell lysates, such as polypeptides produced intracellularly, wherein the host cell is lysed or destroyed to release the polypeptides. Let's do it.

As used herein, the term "metal chelating agent" refers to a molecule that forms a complex of metal ions. By way of example, such molecules form two or more coordination bonds with a central metal ion and optionally form a ring structure.

As used herein, the term “metal-containing moiety” refers to a group containing metal ions, atoms or particles. Such moieties include, but are not limited to, cisplatin, chelated metal ions (eg nickel, iron and platinum) and metal nanoparticles (eg nickel, iron and platinum).

As used herein, the term “residues in which heavy atoms are incorporated” refers to groups in which ions of atoms that are heavier than carbon are incorporated. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead and uranium.

As used herein, the term “modified” refers to the presence of a change to an amino acid or amino acid residue, which change or modification is obtained by a chemical or biochemical process.

As used herein, the term "regulated serum half-life" refers to a positive or negative change in the circulating half-life of a modified biologically active molecule compared to its unmodified form. By way of example, modified biologically active molecules include, but are not limited to, compounds provided herein. As an example, serum half-life is measured by taking blood samples at various time points after administration of a biologically active molecule or modified biologically active molecule, and determining the concentration of that molecule in each sample. The serum half-life can be calculated using the correlation between serum concentration and time. As an example, the regulated serum half-life may be an increase in serum half-life, which allows for improved dosing regimens or to avoid toxic effects. Such increase in serum may be at least about 2 times, at least about 3 times, at least about 5 times, or at least about 10 times.

The term "nanoparticle" as used herein refers to a particle having a particle size of about 500 nm to about 1 nm.

As used herein, the term “approximately-stoichiometric” refers to a molar ratio of the compound participating in the chemical reaction from about 0.75 to about 1.5.

As used herein, the term "non-eukaryotic cell" refers to a non-eukaryotic organism. By way of example, non-eukaryotic organisms belong to the eubacterial phylogenetic domain, including Escherichia coli, Thermus thermophilus), or a brush as a Bacillus stearate Ruth (Bacillus stearothermophilus ), Pseudomonas fluorescens , Pseudomonas aeruginosa aeruginosa , Pseudomonas sp. putida ), but are included in, but not limited to, the archaeological phylogenetic domain, including Methanococcus jannaschii , Methanobacterium thermoautotrophicum , Arcaeoglobus Archideoglobus fulgidus ), Pyrococcus furiosus ), Pyrococcus horikoshii , Aeuropyrum pernix ), or halobacterium , such as Haloferax volcanii ) and halobacterium species NRC-1.

The term “nucleic acid” as used herein refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include (i) analogs of natural nucleotides having properties similar to the reference nucleic acid; (ii) oligonucleotide analogues, such as, but not limited to, PNA (peptidonucleic acid), analogs of DNA used in antisense techniques (phosphothioate, phosphoramidate, etc.); (iii) their conservatively modified variants (such as, but not limited to, degenerate codon substitutions) and complementary and clearly indicated sequences. By way of example, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

As used herein, the term “oxidant” refers to a compound or substance capable of removing electrons from a compound to be oxidized. By way of example, oxidants include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol and oxygen. Various oxidants are suitable for use in the methods and compositions described herein.

As used herein, the term “photoaffinity label” refers to a label having a group on which the label forms a link with a molecule having affinity upon exposure to light. By way of example only, such a connection may be shared or non-shared.

As used herein, the term “photocasing moiety” refers to a group that covalently or non-covalently binds with ions or other molecules upon luminescence at a particular wavelength.

As used herein, the term “photodegradable group” refers to a group that breaks upon exposure to light.

As used herein, the term "photocrosslinker" refers to a compound that is reactive upon exposure to light and comprises two or more functional groups that form covalent or non-covalent bonds with two or more monomers or polymer molecules.

The term “photoisomerization moiety” as used herein refers to a group that changes from one isomeric form to another upon luminescence by light.

The term "polyalkylene glycol" as used herein refers to a linear or branched polymeric polyether polyol. Such polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol and derivatives thereof.

The term "polymer," as used herein, refers to a molecule consisting of repeated subunits. Such molecules include, but are not limited to, proteins, polypeptides, peptides, polynucleotides or polysaccharides or polyalkylene glycols.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. In other words, the description of the polypeptide is equally applicable to the description of the peptide and the description of the protein, and vice versa. Amino acid residues include residues derived from natural and unnatural amino acids. The term applies to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues is a compound provided herein. In addition, such “polypeptides”, “peptides” and “proteins” comprise amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds or other linkages.

The term “post-translationally modified” refers to any modification of an amino acid that occurs after the amino acid is incorporated into the polypeptide chain by translation. Such modifications include, but are not limited to, post-translational in vivo modifications and post-translational in vitro modifications.

As used herein, the term “protected” refers to the presence of “protecting groups” or moieties that prevent the reaction of chemically reactive functional groups under certain reaction conditions. The protecting group will depend on the type of chemically reactive group to be protected. By way of example only, (i) when the chemically reactive group is an amine or hydrazide, the protecting group may be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) when the chemically reactive group is thiol, the protecting group may be orthopyridyldisulfide; (iii) When the chemically reactive group is a carboxylic acid such as butanoic or propionic acid, or a hydroxyl group, the protecting group may be a benzyl or alkyl group such as methyl, ethyl or tert-butyl. By way of example only, the blocker / protecting group is also acetamide, allyloxycarbonyl, allyl ether, benzyl, benzylamine, benzylideamine, benzyl carbamate, benzyl ester, methyl ester, t-butyl ester, St-butyl Ester, 2-alkyl-1,3-oxazoline, dimethyl acetal, 1,3-dioxane, 1,3-dithiane, N, N-dimethylhydrazone, phthalimide, trityl, paramethoxybenzyl ether Carbobenzyloxy, p-toluenesulfonamide, trifluoroacetamide, triphenylmethylamine, t-butyl ether, benzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, 2- (trimethyl Silyl) ethoxycarbonyl, acetic acid ester, pivalic acid ester, benzoic acid ester, acetonide, benzylidene acetal, and photodegradable groups such as Nvoc and MeNvoc.

As used herein, the term “radioactive moiety” refers to a group in which the nucleus spontaneously emits nuclear radiation, such as alpha or beta particles, or gamma radiation.

As used herein, the term "reactive compound" refers to a compound that is reactive to another atom, molecule or compound under appropriate conditions.

The term “recombinant host cell” (also referred to as a “host cell”) refers to a cell comprising an exogenous polynucleotide, wherein methods used to insert the exogenous polynucleotide into a cell include direct uptake, transformation, f-crossing, or Other methods known in the art for the production of recombinant host cells include, but are not limited to. By way of example only, such exogenous polynucleotides may be unincorporated vectors, such as but not limited to plasmids, or may be incorporated into the host genome.

As used herein, the term "redox-active agent" refers to a molecule whose redox activator is reduced or oxidized by oxidizing or reducing another molecule. Examples of redox activators include, but are not limited to, ferrocene, quinone, Ru 2 + / 3 + complexes, Co 2 + / 3 + complexes, and Os 2 + / 3 + complexes.

As used herein, the term “reducing agent” refers to a compound or substance that can add an electron to a compound to be reduced. By way of example, reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol) and reduced glutathione. By way of example only, such reducing agents can be used to maintain sulfhydryl groups in the reduced state and to reduce intramolecular or intermolecular disulfide bonds.

As used herein, the term “refolding” describes any process, reaction or method of modifying an improperly folded or unfolded state into its original or appropriately folded form. By way of example only, refolding with respect to disulfide bonds is the modification of disulfide bonds containing a protein or polypeptide to their original or appropriately folded form in an improperly folded or unfolded state. Such disulfide bonds containing proteins or polypeptides include proteins or polypeptides in which a compound provided herein is incorporated. Refolding is often initiated by the removal of chaotropic agents, such as urea or guanidinium hydrochloride, which have been previously added to the protein solution for solubilization and unfolding of the protein.

As used herein, the term “resin” refers to high molecular weight, insoluble polymer beads. By way of example only, such beads can be used as a support for solid phase peptide synthesis or as a site for attachment of molecules prior to purification.

As used herein, the term “saccharide” refers to a series of carbohydrates such as, but not limited to, sugars, monosaccharides, oligosaccharides and polysaccharides.

As used herein, the term “spin label” is an atom or group of atoms representing an unpaired electron spin that can be detected by electron spin resonance spectroscopy and attached to another molecule (ie, a stable paramagnetic group). It refers to a molecule containing. Such spin-labeled molecules include, but are not limited to, nitrile radicals and nitroxides, and may be single spin labels or dual spin labels.

As used herein, the term “stoichiometric” refers to a molar ratio of the compounds participating in the chemical reaction is from about 0.9 to about 1.1.

As used herein, the term “substantially purified” refers to the ingredient of interest that is substantially or essentially free of other ingredients that normally accompany or interact with the ingredient of interest prior to purification. By way of example only, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3% of the components of interest If it contains less than about 2% or less than about 1% (dry weight) of contaminating components, the component of interest may be "substantially purified". Thus, the "substantially purified" component of interest is about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% Or higher purity levels. By way of example only, proteins, polypeptides or peptides containing a compound provided herein are purified from the original cell or host cell. By way of example only, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5% of a preparation of a protein, polypeptide or peptide containing a compound provided herein If it contains less than about 4%, less than about 3%, less than about 2% or less than about 1% (dry weight), the preparation is "substantially purified." By way of example only, when a protein, polypeptide or peptide containing a compound provided herein is produced recombinantly by a host cell, the protein, polypeptide or peptide containing a compound provided herein is about 30% of the dry weight of the cell. %, About 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less. By way of example only, when a protein, polypeptide or peptide containing a compound provided herein is produced recombinantly by a host cell, the protein, polypeptide or peptide containing a compound provided herein is about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, About 50 mg / L, about 10 mg / L or about 1 mg / L or less. By way of example only, “substantially purified” proteins, polypeptides or peptides containing a compound provided herein are suitable methods including, but not limited to, SDS / PAGE analysis, RP-HPLC, SEC and capillary electrophoresis. Measured by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85 Purity level of about 90%, about 95%, about 99% or more.

As used herein, the term "toxic moiety" refers to a compound that can cause harm or cause death.

As used herein, the terms “treat”, “treating” or “treatment” alleviate, reduce or ameliorate the symptoms of a disease or condition, prevent further symptoms, ameliorate or prevent the underlying metabolic cause of symptoms, , Inhibiting a disease or condition, for example, arresting the progression of a disease or condition, alleviating a disease or condition, regressing a disease or condition, or reducing a condition caused by a disease or condition, Stopping the symptoms of the disease or condition. The term “treat”, “treating” or “treatment” includes, but is not limited to, prophylactic and / or curative treatment.

As used herein, the term “water soluble polymer” refers to any polymer that is soluble in an aqueous solvent. Such water soluble polymers include polyethylene glycol, polyethylene glycol propionaldehyde, mono C 1 -C 10 alkoxy or aryloxy derivatives thereof, monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic acid Anhydrides, N- (2-hydroxypropyl) -methacrylamide, dextran, dextran derivatives such as dextran sulfate, polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols, Heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives such as methylcellulose and carboxymethyl cellulose, such as serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives thereof, polyalkyl Copolymers of ethylene glycol and its derivatives, polyvinyl ethyl ether and alpha- Beta-poly [(2-hydroxyethyl) -DL-aspartamide and the like or mixtures thereof, including but not limited to. By way of example only, by the coupling of such a water soluble polymer with a protein, polypeptide or peptide containing a compound provided herein, increased water solubility, increased or regulated serum half-life, therapeutic half-life increased or controlled relative to the unmodified form , Increased bioavailability, regulated biological activity, extended circulation time, regulated immunogenicity, regulated physical association properties such as aggregation and multimer formation, altered receptor binding, altered binding with one or more binding partners, and altered receptor Changes occur, including but not limited to dimerization or multimerization, and the like.

Other objects, features and advantages of the methods, compositions and combinations described herein will become apparent from the following detailed description. It is to be understood, however, that the present description and specific examples show specific embodiments, which are provided by way of illustration only.

Biosynthetically  The resulting pyrrolysine and PCL Site specific incorporation of

Pyrrolysine (PYL) is the 22nd natural genetically encoded amino acid found in certain methanogenic archaea of the Methanosarcinaceae family and two unrelated bacterial species. Specifically, pyrrolysine is found in MtmB1, a monomethylamine (MMA) methyltransferase that initiates the methane formation of these archaea bacteria (Srinivasan, G., James, CM, and Krzycki, JA (2002), " Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA, " Science , 296 , 1459-62]; Soares, JA, Zhang, L., Pitsch, RL, Kleinholz, NM, Jones, RB, Wolff, JJ, Amster, J., Green-Church, KB, and Krzycki, JA (2005), "The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases," Journal of Biological Chemistry , 280 , 36962-9; Hao, B., Gong, W., Ferguson, TK, James, CM, Krzycki, JA, and Chan, MK, (2002), "A new UAG-encoded residue in the structure of a methanogen methyltransferase", Science , 296 , 1462-6; Krzycki, JA, (2005), "The direct genetic encoding of pyrrolysine," Current Opinion in Microbiology , 8 , 706-12; Krzycki, JA, (2004), "Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases," Current Opinion in Chemical Biology , 8 , 484-91, and Ambrogelly, A., Palioura, S., and Soll, D., (2007), "Natural expansion of the genetic code," Nature Chemical Biology 3 , 29-35). Pyrrolysine is believed to be a dipeptide in which the ε-amine of lysine is linked to the D-isomer of 4-methyl-pyrroline-5-carboxylate via an amide bond (Polycarpo, CR, Herring, S., Berube, A., Wood, JL, Soll, D., and Ambrogelly, A., (2006), "Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase," FEBS Letters , 580 , 6695-700). The structure of pyrrolysine (FIG. 1) was inferred from the crystal structure of MtmB1 and the mass of residues (see J. Biol. Chem. 2005, 44, 36962-36969; PNAS, 2007, 104, 1021-1026). ).

The mtmB1 gene encoding MtmB1 normally has an in-frame amber (TAG) codon, which is a reference termination codon. However, in mtmB1 mRNA, UAG codons encoded as TAG at the DNA level do not terminate translation during the production of MtmB1 protein, but instead UAG codons encode pyrrolysine incorporated into the protein. Pyrrolysine is synthesized endogenously and then incorporated into this in-frame UAG codon as a free amino acid by simultaneous translation.

Pyrrolysine is readily biosynthesized and incorporated by the native genes pylT, pylS, pylB, pylC and pylD. pylT encodes pyrrolysyl-tRNA and pylS encodes pyrrolysyl-tRNA synthetase, while pylB, pylC and pylD encode proteins required for biosynthesis of pyrrolysine. These genes are derived from Methanosarcina mazei (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A., Zhang, L., Green-Church, KB, and Krzycki, JA , (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6, and Namy, O., Zhou, Y., Gundllapalli, S., Polycarpo, CR, Denise, A., Rousset, JP, Soll, D., and Ambrogelly, A., (2007), "Adding pyrrolysine to the Escherichia coli genetic code," FEBS Letters , 581 , 5282-8). The pylT and pylS genes together with the pylB, pylC and pylD genes form a pylTSBCD gene cluster, which is a natural genetic code expansion cassette whose delivery is due to the pyrrolysine (endogenously synthesized) in which the UAG codon is incorporated into the protein at the UAG site. Free amino acid).

Biosynthesis of pyrrolysine has been proposed as being facilitated by the gene products of the native genes pylB, pylC and pylD with D-ornithine proposed as precursor (Namy, O., Zhou, Y., Gundllapalli, S., Polycarpo, CR, Denise, A., Rousset, JP, Soll, D., and Ambrogelly, A., (2007), "Adding pyrrolysine to the Escherichia coli genetic code," FEBS Letters , 581 , 5282-8). Various precursors to pyrrolysine, such as D-glutamate, D-isoleucine, D-proline and D-ornithine, have been proposed, but D-ornithine is by means of a plasmid with the native genes pylT, pylS, pylB, pylC and pylD It has been suggested to be the most effective precursor for pyrrolysine biosynthesis in transformed Escherichia coli. This study used overtranslation in the TAG incorporation or breakdown signal, ie the generation of full-length proteins, and in Escherichia coli, where pyrrolysine was transformed by plasmids with the native genes pylT, pylS, pylB, pylC, and pylD And biosynthesis and incorporation into the resulting protein using D-ornithine as precursor. Although incorporation of pyrrolysine was not directly verified by mass spectrometry, the conclusion was that plasmids with the native genes pylT, pylS, pylB, pylC and pylD, although low levels of pyrrolysine in the absence of added D-ornithine It was supported by previous mass spectrometry data in that it was biosynthesized and incorporated into proteins produced in Escherichia coli transformed by (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A. , Zhang, L., Green-Church, KB, and Krzycki, JA, (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6).

However, as provided herein, incorporation of pylT, pylS, pylB, pylC, and pylD genes into Escherichia coli or mammalian cells, as identified using mass spectrometry, and D-ornithes into growth media By the addition of chitin it was found that "demethylated pyrrolysine" is biosynthesized and incorporated (Figure 1, PCL-A and PCL-B). This observation is surprising and unpredictable in light of the results proposed by Longstaff et al. Thus, using the native genes pylT, pylS, pylB, pylC and pylD and D-ornithine as precursors, pyrrolysine analogs, pyrroline-carboxy- naturally encoded, biosynthetically produced and incorporated into proteins Lysine (PCL) is provided herein. In another embodiment, since D-arginine is a precursor to D-ornithine, the naturally occurring genes pylT, pylS, pylB, pylC and pylD and D-arginine as precursors are naturally encoded and biosynthetically produced. Pyrrolysine analogs incorporated into proteins are also provided herein.

Initially, the formation of the pyrroline ring of pyrrolysine from D-ornithine was thought to be similar to the formation of proline from L-ornithine (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A., Zhang, L., Green-Church, KB, and Krzycki, JA, (2007), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6). Numerous bacteria convert L-ornithine to L-proline via the 1-pyrroline-5-carboxylate intermediate. Formation of 1-pyrroline-5-carboxylate from ornithine is the first step in a two step process in the biosynthesis of L-proline. Formation of 1-pyrroline-5-carboxylate from L-ornithine can occur via two pathways, the first route being 1-pyrroline-5-carr by cyclotalaminoation of L-ornithine The formation of the carboxylates and the second pathway is the formation of L-glutamate semialdehydes by L-ornithine amino transferase. Glutamate semialdehyde then forms 1-pyrroline-5-carboxylate. L-proline is formed by reduction of 1-pyrroline-5-carboxylate by pyrroline-5-carboxylate reductase.

Recently, homology with L-ornithine amino transferase has not been confirmed in the formation of pyrrolysine in methanogenic archaea. However, this is expected to be related to similar enzymes. Biosynthesis of pyrrolysine is thought to be a consistent process using cell precursors and a consistent action of the products of the pylB, pylC and pylD genes, which biosynthesize pyrrolysine from D-proline via 1-pyrroline-5-carboxylate The proposed scheme for this is shown in FIG. 2A (Longstaff, DG, Larue, RC, Faust, JE, Mahapatra, A., Zhang, L., Green-Church, KB, and Krzycki, JA, (2007). ), "A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine," Proceedings of the National Academy of Sciences of the United States of America , 104 , 1021-6). pylB, pylD and pylD gene products are members of several protein families. PylB has signature residues of the Fe-S radical SAM enzyme family known to mediate radical-catalyzed reactions. In addition, PylB shares some sequence homology with biotin synthase, so it has been suggested that PylB is involved in the formation or methylation of pyrrolysine pyrroline rings. PylD, the pylD gene product, has NADH-binding domains of several families of dehydrogenases, suggesting that PylD is involved in the formation of 1-pyrroline imine bonds. PylC is similar to carbamoylphosphate synthetase and D-alanyl-D-alanine ligase, which is a pyrrolysine amide between lysine and the D-isomer of 4-methyl-1-pyrroline-5-carboxylate. It suggests a role in the formation of bonds. Thus, putative pylB, pylC and pylD gene products have respective similarities to radical SAM proteins, protein forming amino acids and amino acid dehydrogenases, and are therefore believed to participate in similar pathways for the biosynthesis of pyrrolysine.

However, as provided herein, attempts to biosynthesize pyrrolysine in Escherichia coli and HEK293F cells using D-ornithine as precursor do not form pyrrolysine, but rather "demethylated pyrrolysine" (herein Pyrroline-carboxy-lysine (PCL) (also referred to as PCL-A or PCL-B: see FIG. 1). As described herein, biosynthesis of PCL-A or PCL-B does not require the presence of the pylB gene, so a possible scheme for biosynthesis of PCL-A or PCL-B is shown in FIG. 2B. Without wishing to be bound by any particular theory, this possible route is the conversion of D-ornithine to 5-amino-2-oxopentanic acid via endogenous D-amino acid oxidase (EC 1.4.3.3), and 1-blood. Spontaneous cyclization of 5-amino-2-oxopentanoic acid with roline-2-carboxylate and water. This precursor available in most organisms can be converted to D-1-pyrroline-5-carboxylate, possibly by rearrangement of double bonds, with the aid of the enzyme PylD. Ligation of D-1-pyrroline-5-carboxylate with L-lysine to epsilon amine by PylC and ATP will produce pyrroline-carboxy-lysine (PCL: PCL-A). Alternatively, ligation of 1-pyrroline-2-carboxylate can result in PCL-B having the same molecular weight as PCL-A. The observation that both PylD and PylC are necessary for incorporation of PCL, and that PylS is not acceptable as substrate pyrrolysine analogs with sp2 carbon at positions equivalent to C-5 position of the 1-pyrroline ring of PCL (herein It is suggested initially that PCL can be incorporated into the protein primarily in PCL-A form. Without wishing to be bound by any particular theory, demethylated PCL-A or PCL-B is required cofactor (s) in the absence of methyl donor PylB substrate in the presence of D-ornithine added as a result of inactivation of PylB. In the absence or in combination thereof. However, this does not exclude the presence of alternative mechanisms. Indeed, incorporation tests using several intermediates (as presented herein) suggest different biosynthetic pathways of PCL-A or PCL-B than those suggested in FIG. 2B. This alternative route is described herein.

Incorporation of pyrrolysine into proteins in response to UAG codons is facilitated by the native genes pylT and pylS, where UAG translation as pyrrolysine is converted to amber by pyrrolysyl-tRNA synthetase (PylS) into pyrrolysine It was found that it requires aminoacylation of de-coding tRNA pyl (FIG. 3A). The pylT gene encodes a specific natural inhibitor tRNA pyl (PylT), whose CUA anticodons are complementary to the UAG sense codons corresponding to pyrrolysine. The pylS gene encodes a specific class II tRNA synthetase, pyrrolysyl-tRNA synthetase (PylS), which gives tRNA pyl pyrrolysine (either chemically or biosynthetically synthesized) and also pyrrolysine- A dependent ATP: pyrophosphate exchange reaction is performed. Similarly, incorporation of the pyrrolysine analog PCL-A or PCL-B into proteins in response to UAG codons is thought to be facilitated by the native genes pylT and pylS. (FIG. 3B).

Biosynthesis and site-specific incorporation of pyrrolysine and PCL into proteins expressed by prokaryotic and eukaryotic cells

Biosynthetically produced pyrrolysine and / or pyrroline-carboxy-lysine ((S) -2-amino-6- (3,4-dihydro-2H-pyrrole-2-carboxamido) hexanoic acid ( PCL-A) or a method for site specific incorporation of (S) -2-amino-6- (3,4-dihydro-2H-pyrrole-5-carboxamido) hexanoic acid (PCL-B)) Provided herein. Pyrrolysine analogs PCL-A and PCL-B, both referred to herein as PCL, lack the methyl group of pyrrolysine (PYL) (FIG. 1). In certain embodiments of this method, the eukaryotic cell is a mammalian cell, yeast cell, insect cell, fungal cell or plant cell. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamsters. Renal (BHK-21) cells and Chinese hamster ovary (CHO) cells are included, but are not limited to these. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera ( Spodoptera). frugiperda ) (sf9 and sf21) cells, Trichoplusia ni (BTI TN-5B1-4 or High-Five ™) cells and Mammestra Brassica ( Mammestra) brassicae ) cells, including but not limited to. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, the bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis ( Mycobacterium). smegmatis ), Lactococcus lactis ) and Bacillus subtilis ), but are not limited thereto.

In certain embodiments, such methods for site-specific incorporation of biosynthetically produced pyrrolysine and PCL include the genes pylT, pylS, pylB, pylC and pylD, and genes for the desired proteins in prokaryotic and / or eukaryotic Incorporating cells and optionally adding precursors for pyrrolysine or PCL to the growth medium of the transfected cells. In certain embodiments, the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D, L-ornithine. In certain embodiments, the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D, L-arginine. In certain embodiments, the precursor is

Figure pat00043
: (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the precursor is
Figure pat00044
: (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the precursor is
Figure pat00045
2,5-diamino-3-methylpentanoic acid. In certain embodiments, the precursor is (2R, 3R) -2,5-diamino-3-methylpentanoic acid. In certain embodiments, the eukaryotic cells are mammalian cells, yeast cells, insect cells, fungal cells or plant cells. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited to these. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells and Mammastra Brassica. Include but are not limited to cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, the bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis. However, the present invention is not limited thereto.

In certain embodiments, such methods for site-specific incorporation of biosynthetically produced pyrrolysine and PCL include the genes pylT, pylS, pylB, pylC and pylD, and genes for the desired proteins in prokaryotic and / or eukaryotic Incorporation into cells and the addition of precursors for pyrrolysine or PCL to the growth medium of the transfected cells. In certain embodiments, the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D, L-ornithine. In certain embodiments, the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D, L-arginine. In certain embodiments, the precursor is (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the precursor is (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the precursor is 2,5-diamino-3-methylpentanoic acid. In certain embodiments, the precursor is (2R, 3R) -2,5-diamino-3-methylpentanoic acid. In certain embodiments, the eukaryotic cells are mammalian cells, yeast cells, insect cells, fungal cells or plant cells. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited to these. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells and Mammastra Brassica. Include but are not limited to cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, the bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis. However, the present invention is not limited thereto.

In certain embodiments, such methods for site specific incorporation of biosynthetically generated PCL include incorporating genes pylT, pylS, pylC and pylD, and genes for the desired protein into prokaryotic and / or eukaryotic cells, and growing Adding D-ornithine as a precursor for PCL to the medium. In certain embodiments, the eukaryotic cells are mammalian cells, yeast cells, insect cells, fungal cells or plant cells. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited to these. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells and Mammastra Brassica. Include but are not limited to cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, the bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis. However, the present invention is not limited thereto. In these embodiments where the native genes pylT, pylS, pylC and pylD are used, rather than biosynthesically being produced and incorporated, demethylated pyrrolysine analog PCL is biosynthetically produced and incorporated instead.

In certain embodiments, such methods for site specific incorporation of biosynthetically produced pyrrolysine and PCL include the genes pylT, pylS, pylC and pylD, and genes for the desired proteins in prokaryotic and / or eukaryotic cells. Incorporate D-ornithine, L-ornithine, D, L-ornithine, D-arginine, L-arginine, D, L-arginine, (2S) as precursors for pyrrolysine and / or PCL in the growth medium. ) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid, (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid or Adding 2,5-diamino-3-methylpentanoic acid or (2R, 3R) -2,5-diamino-3-methylpentanoic acid. In certain embodiments, the eukaryotic cells are mammalian cells, yeast cells, insect cells, fungal cells or plant cells. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited to these. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells and Mammastra Brassica. Include but are not limited to cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, the bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis. However, the present invention is not limited thereto. In these embodiments where the native genes pylT, pylS, pylC and pylD are used, rather than biosynthesically being produced and incorporated, demethylated pyrrolysine analog PCL is biosynthetically produced and incorporated instead.

In certain embodiments, such methods for site specific incorporation of biosynthetically produced pyrrolysine include incorporating the genes pylT, pylS, pylC and pylD, and genes for the desired protein into prokaryotic and / or eukaryotic cells, Adding 2,5-diamino-3-methylpentanoic acid or (2R, 3R) -2,5-diamino-3-methylpentanoic acid as precursors for pyrrolysine to the growth medium. In certain embodiments, the eukaryotic cells are mammalian cells, yeast cells, insect cells, fungal cells or plant cells. In other embodiments, the mammalian cells used in the methods provided herein include human embryonic kidney HEK293F cells, human epithelial carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK-21 cells, and Chinese hamster ovary CHO cells are included, but are not limited to these. In certain embodiments, yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells and Mammastra Brassica. Include but are not limited to cells. In certain embodiments, the prokaryotic cells are bacteria, while in other embodiments, the bacteria used in the methods provided herein include Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis. However, the present invention is not limited thereto.

Site specific incorporation of biosynthetically generated PCL at the TAG encoded site of the model protein (hRBP4) utilizes the native genes pylT, pylS, pylB, pylC and pylD, and D-ornithine added to the growth medium as precursors Was performed in mammalian cells (see Example 2). In one embodiment, site specific incorporation of PCL into the model protein, hRBP4, allows HEK293F cells to be UAG-recognized tRNA PylT, DNAs encoding their specific aminoacyl-tRNA synthetase PylS, biosynthetic genes pylB, pylC and pylD, This was accomplished by cotransfection with DNA encoding the target protein with the incorporation site encoded by TAG. Another gene construct used for such in vivo biosynthesis and incorporation in mammalian cells into the model protein hRBP4 at a single site specified by the TAG codon is shown in FIG. 4A. Other gene constructs for use in mammals for the expression of hRBP4, mEPO, hEPO, and various single site TAG mutants of the Fc domain of mIgG1 are shown in FIG. 4B. Plasmids with pylT, pylS, pylB, pylC and pylD for incorporation of biosynthetically induced PCL or pyrrolysine in Escherichia coli cells are shown in FIG. 5.

D-ornithine, the putative precursor for the biosynthesis of pyrrolysine, is added to the culture medium of HEK293F cells transfected with DNA encoding the target protein as well as pylT, pylS, pylB, pylC and pylD, but not PCL. It is incorporated into hRBP4 at the site of this TAG codon (see Example 2, FIG. 6A). The incorporation efficiency depends on the different incorporation sites as shown in Figure 6A.

Site-specific incorporation of biosynthetically generated PCL at the TAG encoded site of the model protein (hRBP4) is achieved in mammals using the native genes pylT, pylS, pylC and pylD, and D-ornithine added to the growth medium as precursors. Carried out in cells (see Example 2). In one embodiment, site specific incorporation of the PCL into the model protein, hRBP4, causes HEK293F cells to be incorporated into TAG, as well as DNA encoding UAG-recognized tRNA PylT, its specific aminoacyl-tRNA synthetase PylS, biosynthetic genes pylC and pylD. By co-transfection with DNA encoding a target protein having an incorporation site encoded by When D-ornithine, a putative precursor for the biosynthesis of pyrrolysine, is added to the culture medium of transfected HEK293F cells, PCL, not pyrrolysine, is incorporated into hRBP4 at the site of the TAG codon. FIG. 6B shows SDS-PAGE and FIG. 6C shows purified hRBP4 Phe62PCL (mutant # 2) produced in HEK293F cells in the absence (B, lane 1) or presence (D, or 2) of D-ornithine The mass spectrum is shown. The arrow indicates the full length hRBP4 and the mass obtained was 23166.0 Da, which is close to the expected mass 23168 Da. In addition, the mass spectrometry data shown in FIGS. 7-9 show that PCL, not pyrrolysine, is incorporated at the TAG site at residue 62 of hRBP4 (see Example 2).

1 depicts the structure (structure PCL-A or alternative structure PCL-B) of pyrrolysine (PYL) and demethylated pyrrolysine analogues, pyrroline-carboxy-lysine (PCL). The structure of PCL-A (or alternative structure PCL-B) was distinguished from pyrrolysine using high precision mass spectrometric analysis of peptide fragments at the site of incorporation (FIGS. 7-9). In addition, the structure of PCL was distinguished from pyrrolysine by detection of PCL by mass spectrometry as a free amino acid in cell lysates (see FIG. 10, Example 3). In addition, observation of PCL in cell lysates demonstrates that PCL is biosynthetically produced as free amino acid, rather than formed by post-translational modifications. 10A and 10C show the detection of PCL in lysates from HEK293F cells biosynthesized from D-ornithine. 10A is an HPLC trace of lysates from cells transfected with the biosynthetic genes pylB , pylC and pylD and grown in the presence of D-ornithine (bottom trace) and in the absence of D-ornithine (top trace). The lysate chromatogram obtained from cells grown in the presence of D-ornithine is characterized by a peak at 4.13 min elution time (indicated by an asterisk), but in lysates of the same cells cultured in the absence of D-ornithine does not exist. 10C is the full scan mass spectrum of the HPLC peak at 4.13 min, where the mass (m + 1) obtained is consistent with the theoretical mass for PCL. FIG. 10B is an HPLC chromatogram showing that lysine is equally abundant in both samples, and the total scanning mass (m + 1) spectrum of lysine HPLC at 1.44 minutes illustrates the accuracy of the method (FIG. 10D).

11 depicts incorporation of N-ε-cyclopentyloxycarbonyl-L-lysine (CYC) into various hRBP4 TAG mutant proteins in HEK293F cells. 12 shows mass spectrometric verification of CYC incorporation at the TAG site of hRBP4 mutant Phe62TAG. 13A and 13B (see Example 4) illustrate PCL incorporation as a function of the various precursors listed in the table and shown in FIG. 14A. 13A and 13B also show the direct incorporation of various pyrrolysine analogs (including CYC) into hRBP4 TAG mutant proteins using HEK293F cells. To demonstrate that D-ornithine is a precursor for the biosynthesis of PCL, potential precursors for PCL (FIG. 14A) were selected from the pyrrolysine biosynthetic pylB, pylC and pylD genes, pylT / pylS tRNA / pyrrolysyl-tRNA synthetase. Paired and added to the growth medium of HEK293F cells transfected with hRBP4 TAG mutant DNA. In addition, various pyrrolysine analogs added to the growth medium of HEK293F cells transfected with only pylT / pylS tRNA / aa-tRNA synthetase pairs and hRBP4 TAG mutant DNA are shown in FIG. 14B. FIG. 13A is a Western blot of full length hRBP4 protein with anti-His tag antibody, while FIG. 13B is SDS-PAGE of the same sample after Ni-NTA purification. Although D-arginine (lane 4), which can be metabolized to D-ornithine, exceeds background protein production, as shown, D-ornithine (lane 2) is a better precursor for PCL biosynthesis. Of the pyrrolysine analogues, only CYC produces proteins at levels similar to D-ornithine, whereas the 3-oxobutanoic acid analogue TU3000-016 shows measurable but very low incorporation. Techniques for synthesizing the various analogs are provided in Example 33. All lanes show the production of low levels of full-length protein, possibly due to low endogenous levels of D-ornithine and metabolites or acceptable media components as PylS substrates.

Incorporation of PCL was assessed using different combinations of biosynthetic genes pylB, pylC and pylD (FIG. 13C). The data show that only genes pylC and pylD are essential for PCL biosynthesis and subsequent protein incorporation. Most notably D-proline, other D-proline analogs and D-glutamic acid (FIGS. 13 and 14) did not show full-length protein production above background. This suggests that the biosynthesis of PCL-A and PCL-B does not follow the route suggested in FIG. 2A. However, incorporation tests using 3,4-dihydro-2H-pyrrole-5-carboxylate (P2C) and 1-pyrroline-5-carboxylate (P5C) also produce full-length proteins in Escherichia coli. On the other hand, both synthetic PCL-A and PCL-B were incorporated with high efficiency (FIG. 15A, Example 15). Thus, the biosynthetic pathway proposed in FIG. 2B may also not be a promising pathway.

By incorporation of the precursor (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid (also referred to as Lys-Nε-D-Orn), a significant amount of full-length protein Obtained (FIG. 15A, Example 15). In addition, incorporation of (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid was found to require only the presence of the pylS, pylT and PylD genes (FIG. 15B). , Example 15). Together, this observation suggests an alternative route shown in FIGS. 16A and 16B. This pathway is characterized by the fact that D-ornithine is first coupled to the epsilon amino group of L-lysine with the help of PylC, the putative D-alanyl-D-alanine ligase, (2S) -2-amino-6-(( R) -2,5-diaminopentaneamido) hexanoic acid. In FIG. 16A, the (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid intermediate is D-ornithine: oxygen oxidoreductase (EC 1.4.3.3) or Activated by D-amino-transaminase (EC 2.6.1.21) to form PCL-B by spontaneous cyclization. Alternatively, in FIG. 16B the (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid intermediate is D-ornithine: 2-oxidogglutarate 5- Activated by transaminase (EC 2.6.1.13) to form PCL-A by spontaneous cyclization. Both cyclization reactions are similar to those of D-glutamate 5-semialdehyde. One of the activation steps before cyclization can be presumably catalyzed by PylD and the second step requires an enzyme for the biosynthesis of PCL. As shown in FIG. 16, it is suggested that methylation of PCL-A by PylB completes biosynthesis of PYL. Note that in the case of the pathway of FIG. 16A, it is likely that this would require the presence of another Pyl enzyme to be found that forms PCL-A from PCL-B. However, this additional enzyme would not require the alternative route shown in 16B. PCL-A formed after activation and spontaneous cyclization by PylD can be methylated directly by PylB to form pyrrolysine. Alternatively, any intermediate or D-ornithine may be a substrate for methylation by PylB if subsequent enzymes tolerate this modification of the substrate.

Pyrrolysyl-tRNA synthetase PylS has been shown to allow several pyrrolysine analogs to confer tRNA Pyl . However, this study also pointed out the importance of the pyrrolysine ring C-5 stereocenter for the recognition of synthetase PylS, where the D-form is required (Polycarpo, CR, Herring, S., Berube, A., Wood, JL, Soll, D., and Ambrogelly, A., (2006), "Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase," FEBS Letters , 580 , 6695-700). As provided herein, in line with this interpretation, attempts to incorporate various pyrrolysine analogs, including aromatic 5 and 6-membered ring pyrrolysine analogs (FIG. 13), are directed to such aromatic 5 and 6-membered ring analogs. Proved to not be an acceptable substrate for PylS (FIG. 14). This is probably due to the lack of C-5 chiral centers or, in certain cases, due to larger sized analogs. As indicated, N-ε-cyclopentyloxycarbonyl-L-lysine (CYC), a known substrate for PylS, and TU3000-016, a 3-oxobutanoic acid analog, are PylT / PylS tRNA / pyrrolysyl- Incorporation was done using tRNA synthetase pairs (FIG. 14). However, analogs similar in size and bulk to N-ε-cyclopentyloxycarbonyl-L-lysine (CYC) were evaluated to have not been incorporated because they possibly lacked the C-5 stereocenter. Thus, PylS appears to be selective depending on chirality at the point of attachment of the pyrroline ring. However, synthetic PCL-B was also incorporated with high efficiency, suggesting that sp2 achiral carbon at the C-5 position does not favor incorporation in all cases. Currently, the low reactivity of synthetic PCL-B with 2-ABA compared to synthetic PCL-A (Examples 44, FIGS. 56 and 58), and the known enzymes for the conversion of PCL-B to PCL-A in FIG. 16A Absence favors assignment of pyrrolysine analogs incorporated as PCL-A.

As provided herein, in certain embodiments, biosynthesis of pyrrolysine in mammalian cells (Example 2) and bacterial cells (Examples 8, 9 and 10) when D-ornithine is added to the growth medium as a precursor The use of the native genes pylB, pylC and pylD for the production of PCL rather than pyrrolysine has been shown. In addition, evaluation of the biosynthesis of PCL using native genes pylB and pylC, native genes pylB and pylD, or native genes pylC and pylD produced PCL only when pylC and pylD gene products were present. This suggests that when D-ornithine is added to the growth medium as a precursor, the gene product of pylB is not required for the biosynthesis of PCL (see Figure 13C, Example 4). This was done without co-transfection of gene pylB into mammalian cells, three model proteins, hRBP4 (see FIG. 6, Example 2), mIgG1 Fc domain (FIG. 17A, see Example 5) and mEPO (FIG. 17B, Supported by the incorporation of PCL). In Escherichia coli, FAS-TE, FGF21 and FKBP are examples of the production of PCL exclusively using the native genes pylB, pylC and pylD (Figures 18B, 19 and 20, Examples 8, 9 and 10).

Biosynthesis of PCL requires the presence of biosynthetic genes pylC and pylD but not pylB in the host cell. In the biosynthesis of pyrrolysine in methasorcina, it is understood that PylD contains the NADH-binding domain of dehydrogenase, thus producing D-1-pyrroline-5-carboxylate from D-proline Proposed. However, adding D-proline to the growth medium does not show significant PCL incorporation (FIG. 13). In addition, 3,4-dihydro-2H-pyrrole-5-carboxylate (also referred to herein as 1-pyrroline-2-carboxylate; also referred to as P2C) and D-1-pyrroline-5- Addition of carboxylate (P5C) failed to produce full length protein, while (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid produced PCL containing protein do. PylC has sequence homology with D-alanyl-D-alanine ligase and catalyzes the attachment of D-ornithine to the epsilon-amino group of lysine in biosynthesis of PCL or pyrrolysine (2S) -2-amino- 6-((R) -2,5-diaminopentaneamido) hexanoic acid may be provided. Thus, it is not bound to any theory herein, and the biosynthesis of PCL from D-ornithine in mammalian or Escherichia coli cells is characterized by the (2S) -2-amino-6- of D-ornithine by PylC. It is assumed that it may be related to the conversion to ((R) -2,5-diaminopentaneamido) hexanoic acid (FIG. 16B). As shown in FIG. 16B, PylD is a semialdehyde ((S) -2-(-6S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid spontaneously cyclized to PCL-A. ) -2-amino-6-((R) -2-amino-5-oxopentanamido) hexanoic acid). Alternatively, PCL-B will form spontaneously if PylD has D-amino acid transaminase or D-ornithine: oxygen oxidoreductase-like activity (FIG. 16A).

In other embodiments, expression of mouse EGF and mouse TNF-α in Escherichia coli (FIGS. 21 and Examples 13 and 14) results in a protein mixture with PCL or pyrrolysine incorporated at the TAG site. Incorporation of pyrrolysine was dependent on the presence of the pylB gene, but homogeneous preparations of PCL containing proteins were observed in the absence of pylB (Figure 21 and Examples 11 and 12). Thus, this experimentally demonstrates PylB as methyltransferase in the biosynthesis of pyrrolysine. Even in the presence of the pylB gene, proteins containing relative amounts of PCL and pyrrolysine typically vary from fermentation to more promising PCL proteins. This observation suggests that methyltransferase activity or methyl-donoring substrates or necessary cofactors are limited for efficient pyrrolysine biosynthesis in Escherichia coli and mammalian cells.

In addition, the gene product of pylB may not be efficiently expressed. Thus, in certain embodiments, the modified pylB gene is used for biosynthesis of pyrrolysine or other pyrrolysine analogs. Accordingly, provided herein are methods for biosynthesis of pyrrolysine, PCL and other pyrrolysine analogs in Escherichia coli, mammals and other host cells, wherein one or more of the pylB, pylC and pylD genes have been modified. Such modifications may include using other organisms, such as, but not limited to, homologous genes from other species or mutated genes to Metanosarcine. In certain embodiments site directed mutagenesis is used, while in other embodiments random mutagenesis in combination with selection is used. This method also includes adding the DNA of the desired protein and incorporating the pylT and pylS genes to incorporate pyrrolysine, PCL or pyrrolysine analogs into the protein. In certain embodiments, modified pylB genes, native pylC, pylD, pylT and pylS genes are used to biosynthesize and incorporate pyrrolysine. In other embodiments, modified pylB and pylC genes, native pylD, pylT and pylS genes are used to biosynthesize and incorporate pyrrolysine analogs other than PCL. In other embodiments, modified pylB and pylD genes, native pylC, pylT and pylS genes are used to biosynthesize and incorporate pyrrolysine analogs other than PCL. In other embodiments, the pylT, pylS, pylB, pylC and pylD genes are optionally modified to improve incorporation of pyrrolysine, PCL or other pyrrolysine analogs into proteins.

In addition, for certain embodiments, the formation of intermediates in the biosynthesis of pyrrolysine, PCL and / or other pyrrolysine analogs from D-ornithine or the biosynthesis of pyrrolysine may be limited by the function of host enzymes and proteins. . In certain embodiments, low activity or concentration of one or more host enzymes may limit the formation of intermediates required for biosynthesis of pyrrolysine, PCL or other pyrrolysine analogs. In certain embodiments, the activity of a host enzyme can convert an intermediate from other pathways leading to pyrrolysine, PCL or other pyrrolysine analogs, or limit the formation of such intermediates. Thus, provided herein are methods for biosynthesis of pyrrolysine, PCL and other pyrrolysine analogs in Escherichia coli, mammalian or other host cells, wherein one or more host enzymes have been modified. Such methods include overexpression, activation, inhibition or inhibition of such host enzymes by genetic or chemical means, addition of DNA encoding such host enzymes, addition of silent RNA (siRNA) to inhibit mRNA translation, and D- Addition of cofactors necessary for the formation of the intermediate from ornithine is included, but is not limited thereto.

Site specific incorporation of biosynthetically generated PCL at the TAG encoded site was also observed at four sites of the Fc domain of mouse IgG1 (see Example 5 and FIG. 17A) and at 11 sites of erythropoietin (EPO) ( Example 6 and FIG. 17B). PCL incorporation for both sets of proteins was achieved using D-ornithine added to the medium as precursor in HEK293F mammalian cells infected with the native genes pylT, pylS, pylC and pylD. FIG. 17A is SDS-PAGE depicting four full length (FL) mFc proteins, where PCL is HEK293F cells and D co-transfected with respective TAG mutant constructs of mFc, pCMVpylT, pCMVpylS, pCMVpylC and pCMVpylD (FIG. 4A) Cells grown in the presence of ornithine were incorporated into four sites of the mouse IgG1 Fc domain. FIG. 17B is SDS-PAGE depicting 11 full-length (FL) mEPO proteins, where PCL is in the presence of D-ornithine and HEK293F cells co-transfected with TAG mutant constructs of mEPO, pCMVpylT, pCMVpylS, pCMVpylC and pCMVpylD The grown cells were incorporated into 11 sites of mouse EPO.

Site specific incorporation of biosynthetically generated PCL at the TAG encoded site was also achieved using Escherichia coli cells (see Example 7). A plasmid encoding pylB, pylC, pylD, pylS and pylT, pARA-pylSTBCD, was constructed (FIG. 6A). Two sites of the thioesterase domain of human fatty acid synthetase (FAS-TE) (see Examples 8 and 18), one site of FKBP-12 (see Examples 9 and 19) and fibroblast growth factor PCL-A (or PCL-B) incorporation at 20 sites of 21 (FGF21) (see Example 10 and FIG. 20) resulted in a second expression of Escherichia coli cells with genes for pARA-pylSTBCD and the protein of interest. This was achieved by transforming with plasmids and adding D-ornithine to the growth medium during protein expression.

FIG. 18 shows SDS-PAGE and mass spectra for PCL incorporation at two sites of thioesterase of human fatty acid synthetase (FAS-TE). FAS-TE Tyr2454PCL was expressed and both soluble and insoluble protein fractions were purified by Ni-NTA. FAS-TE Leu2222PCL / Leu2223Ile was expressed with or without D-ornithine in replication culture. Ni-NTA elution is shown on the gel. The mass obtained for each is consistent with that expected. 19 shows SDS-PAGE and mass spectra for PCL incorporation at one site of FKBP-12. The mass obtained (12085.6 Da) is consistent with that expected (12084 Da) for single site incorporation of PCL. Also, the determination of FKBP12-Ile90PCL is shown in FIG. 20 depicts SDS-PAGE showing the incorporation of PCL into FGF21 at multiple sites.

The incorporation of pyrrolysine (PYL) and PCL was found to be dependent on the presence of the pylB gene in the expression system. FIG. 21A shows SDS-PAGE analysis of Pyl- or PCL-incorporation into mTNF-α by codon (CAA) of Gln21 mutated with TAG termination codon (Example 13). When D-ornithine is present, lanes 2 and 4 show similar protein expression levels, with and without pylB, respectively. Lanes 3 (with pylB) and 5 (without pylB) show that the protein is not expressed in the absence of D-ornithine.

In addition, mEGF Tyr10TAG was expressed in the presence of pylB, pylC, pylD, pylT and pylS genes in Escherichia coli using D-ornithine as precursor (Example 14). Circular MS spectra (FIG. 21C, bottom) suggested a mixture of proteins incorporating PYL and PCL. The MS peak of Pyl-containing mEGF at 7309 Da is approximately six times larger than the peak of PCL-containing mEGF at 7295 Da. The incorporation of PYL and PCL at the TAG position was verified by tandem MS spectra of the N-terminal peptides MNSYPGCPSS ( PCL ) DGYCLNGGVCM (SEQ ID NO: 32) and MNSYPGCPSS ( Pyl ) DGYCLNGGVCM (SEQ ID NO: 33) of the mEGF Tyr10TAG mutant protein. Quantification from the relative precursor mass abundance of the different peptides showed that PYL was 5-10 times more abundant than PCL, which is roughly consistent with the circular mass measurement (FIG. 21C, bottom). When mEGF Tyr10TAG was expressed in the presence of pylB, circular MS spectra suggested only PCL incorporation (FIG. 21C, top).

Similarly, when mTNF-α Gln21TAG was expressed with D-ornithine in the presence of pylB, pylC, pylD, pylT and pylS genes in Escherichia coli, tandem MS showed peptide NH ( Pyl ) VEEQLEWLSQR (peptide) of mTNF Gln21PCL PYL and PCL incorporation was verified in SEQ ID NO: 34) and NH ( PCL ) VEEQLEWLSQR (SEQ ID NO: 35). In contrast to mEGF Tyr10TAG, quantification from the relative precursor mass abundance of the different peptides identified by tandem MS showed that PCL was 7 times richer than PYL in the mTNF-α Gln21TAG protein. mTNF-α Gln21TAG was expressed in the absence of the pylB gene, and quantification from the relative precursor mass abundance of different peptides in tandem MS measurements showed only PCL protein within the dynamic range of the experiment. These experiments show that PYL incorporation is strictly dependent on the presence of pylB, the putative methyltransferase in Pyl biosynthesis. However, the relative ratio of PCL to PYL incorporated into the protein in the presence of the pylB gene appears to vary from protein to protein and from fermentation experiments, and thus may depend on the type of expression vector, growth conditions and / or other properties of the host cell culture. .

Provided herein are amino acids having the structures of Formula (V) and Formula (VI):

(V)

Figure pat00046

&Lt; Formula (VI)

Figure pat00047

Here, the compound of Formula (V) or Formula (VI) is biosynthetically produced in a cell comprising a pylB gene, a pylC gene, and a pylD gene, and the cell is in contact with a growth medium containing a precursor. In another embodiment, the compound of Formula (V) or Formula (VI) is biosynthetically produced in a cell comprising a pylC gene and a pylD gene, and the cell is in contact with a growth medium comprising a precursor. In certain embodiments of such biosynthesis, the precursor is ornithine or arginine. In certain embodiments of such biosynthesis, the precursor is D-ornithine or D-arginine. In certain embodiments of this biosynthesis, the precursor is (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid. In another embodiment of this biosynthesis, the precursor is (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid.

As also provided herein, cells to which the compounds of Formulas V and VI are biosynthesized additionally comprise a pylS gene and a pylT gene, wherein the compounds of Formula V or Formula VI comprise aminoacyl tRNA synthetases and one or more selectors of mRNA in the cell. It is incorporated into proteins in cells by tRNAs that recognize codons. Such aminoacyl tRNA synthetase is the gene product of the pylS gene, and tRNA is the gene product of the pylT gene. In certain embodiments, the compound of Formula V or Formula VI is incorporated into a protein in a cell by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is formula V or O-tRNA was aminoacylated by a compound of Formula VI, and the O-tRNA recognized one or more selector codons of mRNA in the cell.

The selector codons for incorporation of pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are amber codons (TAG).

Cells used for biosynthesis and / or incorporation of pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis cells. In other embodiments, eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. Such mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary (CHO) cells include, but are not limited to. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include, but are not limited to, Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells, and Mammstra Brassica cells. It is not limited.

In another aspect provided herein, pyrrolysine, PCL, and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are biosynthesized in feeder cells in contact with a growth medium comprising a precursor, the feeder cells pylB gene, pylC gene and pylD gene. In other embodiments, pyrrolysine, PCL and / or pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are biosynthesized in feeder cells that are in contact with a growth medium comprising a precursor, wherein the feeder cells are pylC gene and pylD Contains genes Such biosynthesized pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are secreted from the feeder cells into the growth medium, whereby they are taken up by the second cell and subsequently the second. It is incorporated into proteins synthesized in cells. This second cell contains the pylS gene and the pylT gene. Pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), may be used in a second cell by aminoacyl tRNA synthetase and a tRNA that recognizes one or more selector codons of mRNA in the second cell. Incorporated into proteins. Such aminoacyl tRNA synthetase is the gene product of the pylS gene, and tRNA is the gene product of the pylT gene. In certain embodiments, the compound of Formula V or Formula VI is incorporated into a protein in a second cell by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is O-tRNA was aminoacylated by V or a compound of Formula VI, and the O-tRNA recognized one or more selector codons of mRNA in the second cell.

In certain embodiments of this biosynthesis using feeder cells, the precursor is ornithine or arginine. In certain embodiments of such biosynthesis, the precursor is D-ornithine or D-arginine. In certain embodiments, the precursor is (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the precursor is (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.

The selector codons for incorporation of biosynthesized pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formulas V and VI, using feeder cells are amber codons (TAG).

In certain embodiments, the second cell is a cell of the same type as the feeder cell, while in other embodiments, the second cell is a different cell type than the feeder cell. The cells used for such biosynthesis and / or incorporation of pyrrolysine, PCL and / or other pyrrolysine analogs, such as the compounds of Formulas V and VI, are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis cells. In other embodiments, eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. Such mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary (CHO) cells include, but are not limited to. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include, but are not limited to, Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells, and Mammstra Brassica cells. It is not limited.

In another aspect provided herein, pyrrolysine, PCL and / or other pyrrolysine analogs such as compounds of Formula (V) and Formula (VI) are biosynthesized in feeder cells, and then such biosynthesized pyrrolysine and / or PCL, such as Compounds of formula (V) and formula (VI) are purified from feeder cell cultures and added to the growth medium of a second culture containing second cells. This purified pyrrolysine, PCL and / or other pyrrolysine analogs are then incorporated into the protein synthesized in the second cell. In certain embodiments, pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are biosynthesized in feeder cells in contact with a growth medium comprising a precursor, wherein the feeder cells are pylB gene, It contains pylC gene and pylD gene. In other embodiments, pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are biosynthesized in feeder cells that are in contact with a growth medium comprising a precursor, wherein the feeder cells comprise the pylC gene and It contains the pylD gene. The second cell used in this aspect contains the pylS gene and pylT gene. Pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), may be used in a second cell by aminoacyl tRNA synthetase and a tRNA that recognizes one or more selector codons of mRNA in the second cell. Incorporated into proteins. Such aminoacyl tRNA synthetase is the gene product of the pylS gene, and tRNA is the gene product of the pylT gene. In certain embodiments, the compound of Formula V or Formula VI is incorporated into a protein in a second cell by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein O-RS is O-tRNA was aminoacylated by V or a compound of Formula VI, and the O-tRNA recognized one or more selector codons of mRNA in the second cell.

In certain embodiments of this biosynthesis using feeder cells, the precursor is ornithine or arginine. In certain embodiments of such biosynthesis, the precursor is D-ornithine or D-arginine. In certain embodiments, the precursor is (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid. In certain embodiments, the precursor is (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.

The selector codons for incorporation of biosynthesized pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formulas V and VI, using feeder cells are amber codons (TAG).

In certain embodiments, the second cell is a cell of the same type as the feeder cell, while in other embodiments, the second cell is a different cell type than the feeder cell. The cells used for such biosynthesis and / or incorporation of pyrrolysine, PCL and / or other pyrrolysine analogs, such as compounds of Formula (V) and Formula (VI), are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis cells. In other embodiments, eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. Such mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary (CHO) cells include, but are not limited to. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include, but are not limited to, Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells, and Mammstra Brassica cells. It is not limited.

Also provided herein are amino acids having the structure of Formula VII:

(VII)

Figure pat00048

Wherein the compound of formula VII, or an isomer or tautomer thereof, is biosynthetically produced in a cell containing the pylB gene, pylC gene and pylD gene, and the cell is either D-ornithine or D-arginine or (2S) -2 -Amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid or 2,5-diamino-3-methylpentanoic acid or (2S) -2-amino-6- (2,5 -Diaminopentaneamido) in contact with a growth medium containing hexanoic acid. In another embodiment, such compounds of Formula (VII) are biosynthetically produced in cells containing the pylC gene and pylD gene, and the cells are contacted with a growth medium containing 2,5-diamino-3-methylpentanoic acid Doing. In certain embodiments, the cells are in contact with a growth medium containing D-2,5-diamino-3-methylpentanoic acid. In certain embodiments, 2,5-diamino-3-methylpentanoic acid is (2R, 3S) -2,5-diamino-3-methylpentanoic acid. In certain embodiments, 2,5-diamino-3-methylpentanoic acid is (2R, 3R) -2,5-diamino-3-methylpentanoic acid.

Cells in which the compound of formula (VII) is biosynthesically further comprises a pylS gene and a pylT gene, wherein the compound of formula (VII) is intracellular by a tRNA that recognizes an aminoacyl tRNA synthetase and one or more selector codons of mRNA in the cell. Is incorporated into the protein. Such aminoacyl tRNA synthetase is the gene product of the pylS gene, and tRNA is the gene product of the pylT gene. In certain embodiments, the compound of Formula VII is incorporated into a protein in a cell by orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS), wherein the O-RS is incorporated by the compound of Formula VII The O-tRNA was aminoacylated and the O-tRNA recognized one or more selector codons of mRNA in the cell.

The selector codon for incorporation of the compound of formula VII is amber codon (TAG).

The cells used for biosynthesis and / or incorporation of the compound of formula VII are prokaryotic or eukaryotic cells. In certain embodiments, prokaryotic cells include, but are not limited to Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis cells. In other embodiments, eukaryotic cells include, but are not limited to, mammalian cells, yeast cells, fungal cells, plant cells, or insect cells. Such mammalian cells include human embryonic kidney (HEK293F) cells, human epithelial carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary (CHO) cells include, but are not limited to. In certain embodiments, yeast cells include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells include, but are not limited to, Spodoptera prugiferda sf9 and sf21 cells, Tricofluciani (BTI TN-5B1-4 or High-Five ™) cells, and Mammstra Brassica cells. It is not limited.

In certain embodiments, one or more pyrrolysine, PCL and / or other pyrrolysine analogs are incorporated into proteins, polypeptides and / or peptides using the methods provided herein, and such pyrrolysine and / or PCL are described herein. Derivatized using the method provided in.

PCL  And pyrrolysine Derivatization

Provided herein are proteins, polypeptides or peptides having the structure according to formula (I)

<Formula I>

Figure pat00049

Where

R 1 is H or an amino terminal modification group;

R 2 is OH or a carboxy terminus modification group;

n is an integer from 1 to 5000;

Each AA is independently selected from an amino acid residue, a residue having the structure of Formula A-1, and a residue having the structure of Formula B-1;

Figure pat00050
, here

R 6 is H or C 1 alkyl and at least one AA is a pyrrolysine or pyrrolysine analog having the structure of Formula A-1 or Formula B-1, or an isomer thereof.

In certain embodiments of such compounds of Formula (I), R 6 is H and the residues having the structure of Formula A-1 and the residues having the structure of Formula B-1 are the structures of Formula A-2 and Formula B-2, respectively Residue, or isomer thereof.

Figure pat00051

Accordingly, also provided herein are proteins, polypeptides or peptides having a structure according to formula (I).

<Formula I>

Figure pat00052

Where

R 1 is H or an amino terminal modification group;

R 2 is OH or a carboxy terminus modification group;

n is an integer from 1 to 5000;

Each AA is independently selected from amino acid residues, residues having the structure of Formula A-2 and residues having the structure of Formula B-2;

At least one AA is a pyrrolysine or pyrrolysine analog, or isomer thereof, having a structure of Formula A-2 or Formula B-2.

In certain embodiments n is an integer from 1 to 4000. In certain embodiments n is an integer from 1 to 3000. In certain embodiments n is an integer from 1 to 2000. In certain embodiments n is an integer from 1 to 1000. In certain embodiments n is an integer from 1 to 700. In certain embodiments n is an integer from 1 to 800. In certain embodiments n is an integer from 1 to 600. In certain embodiments n is an integer from 1 to 500. In certain embodiments n is an integer from 1 to 400. In certain embodiments n is an integer from 1 to 300. In certain embodiments n is an integer from 1 to 200. In certain embodiments n is an integer from 1 to 100. In certain embodiments n is an integer from 1 to 90. In certain embodiments n is an integer from 1 to 80. In certain embodiments n is an integer from 1 to 70. In certain embodiments n is an integer from 1 to 60. In certain embodiments n is an integer from 1 to 50. In certain embodiments n is an integer from 1 to 40. In certain embodiments n is an integer from 1 to 30. In certain embodiments n is an integer from 1 to 20. In certain embodiments n is an integer from 1 to 10. In certain embodiments n is an integer from 1 to 5.

In addition, a protein, polypeptide and / or of formula (I) comprising mixing a protein, polypeptide and / or peptide of formula (I) containing at least one pyrrolysine and / or PCL residue with a reagent having the structure of formula (III) Or provided herein for site specific labeling of peptides.

<Formula III>

Figure pat00053

Where

R 3, R 5 and each R 4 is H, -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene, polyalkylene glycols, poly (ethylene glycol), -O (CR 11 R 12 ) k -, -S (CR 11 R 12) k - , -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C (O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11 -, -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k- , -C (O) NR 11 (CR 11 R 12 ) k- , -NR 11 C (O) (CR 11 R 12 ) k- , wherein each of R 11 and R 12 is independently H, C 1 - 8 alkyl, halo-substituted -C 1-8 alkyl, or hydroxy-substituted -C 1 - 8 is alkyl, k is an integer from 1 to 12;

X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X 2 , and any combination of these (where, p is 1 to 10,000, X 2 is H, C 1 - 8 are selected from alkyl, protecting group or a terminal functional group Im).

Further, Formula I, comprising mixing a protein, polypeptide and / or peptide of Formula I containing at least one pyrrolysine and / or PCL residue with a reagent having the structure of Formula IV and reacting under appropriate conditions Provided herein are methods for site specific labeling of proteins, polypeptides and / or peptides.

(IV)

Figure pat00054

Where

R 5 is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, aryl, heteroaryl, heterocycloalkyl or Cycloalkyl, and -LX 1 ;

A is C 3 -C 8 cycloalkyl, C 3 -C 8 heterocycloalkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, 9-10 membered fused bicyclic ring or 13- a 14-membered fused tricyclic ring, where A is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene, polyalkylene glycols, poly (ethylene glycol), -O (CR 11 R 12 ) k -, -S (CR 11 R 12) k - , -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C (O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11 -, -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k- , -C (O) NR 11 (CR 11 R 12 ) k- , -NR 11 C (O) (CR 11 R 12 ) k- , wherein each of R 11 and R 12 is independently H, C 1 - 8 alkyl, halo-substituted -C 1-8 alkyl, or hydroxy-substituted -C 1 - 8 is alkyl, k is an integer from 1 to 12;

X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X 2 , and any combination of these (where, p is 1 to 10,000, X 2 is H, C 1 - 8 are selected from alkyl, protecting group or a terminal functional group Im).

In certain embodiments k is an integer from 1 to 11. In certain embodiments k is an integer from 1 to 10. In certain embodiments k is an integer from 1 to 9. In certain embodiments k is an integer from 1 to 8. In certain embodiments k is an integer from 1 to 7. In certain embodiments k is an integer from 1 to 6. In certain embodiments k is an integer from 1 to 5. In certain embodiments k is an integer from 1 to 4. In certain embodiments k is an integer from 1 to 3. In certain embodiments k is an integer from 1 to 2.

In certain embodiments p is an integer from 1 to 8000. In certain embodiments p is an integer from 1 to 7000. In certain embodiments p is an integer from 1 to 6000. In certain embodiments p is an integer from 1 to 5000. In certain embodiments p is an integer from 1 to 4000. In certain embodiments p is an integer from 1 to 3000. In certain embodiments p is an integer from 1 to 2000. In certain embodiments p is an integer from 1 to 1000. In certain embodiments p is an integer from 1 to 500. In certain embodiments p is an integer from 1 to 400. In certain embodiments p is an integer from 1 to 300. In certain embodiments p is an integer from 1 to 200. In certain embodiments p is an integer from 1 to 100. In certain embodiments p is an integer from 1 to 90. In certain embodiments p is an integer from 1 to 80. In certain embodiments p is an integer from 1 to 70. In certain embodiments p is an integer from 1 to 60. In certain embodiments p is an integer from 1 to 50. In certain embodiments p is an integer from 1 to 40. In certain embodiments p is an integer from 1 to 30. In certain embodiments p is an integer from 1 to 20. In certain embodiments p is an integer from 1 to 10. In certain embodiments p is an integer from 1 to 5.

Non-limiting examples of compounds of Formula IV include:

Figure pat00055

Figure pat00056

Figure pat00057

Wherein the compound having at least one polyethylene glycol (PEG) moiety has an average molecular weight ranging from 1000 Da to 50 kDa, where n is 20 to 1200 and exPADRE is

Figure pat00058
And PADRE is
Figure pat00059
And BG1 is
Figure pat00060
And BG2 is
Figure pat00061
Where * represents a phosphothioate linkage.

In certain embodiments proteins, polypeptides and / or peptides containing one or more pyrrolysine and / or PCL derivatized using the methods provided herein have a structure of Formula II:

&Lt;

Figure pat00062

Where

R 1 is H or an amino terminal modification group;

R 2 is OH or a carboxy terminus modification group;

n is an integer from 1 to 5000;

Each BB is an amino acid residue, a pyrrolysine analogue amino acid having a structure of formula A-2, a pyrrolysine analog amino acid residue having a structure of formula B-2, a pyrrolyl having a structure of formula C-1 New analogue amino acid residues, pyrrolysine analogues having the structure of Formula D-1. New analogue amino acid residues, pyrrolysine analogues having the structure of Formula E-1. Amino acid residue, pyrrolysine analogue amino acid having a structure of formula G-1, pyrrolysine analog amino acid residue having a structure of formula H-1, pyrrolysine analog amino acid residue of structure of formula I-1 , A pyrrolysine analog amino acid residue having the structure of Formula J-1, a pyrrolysine analog amino acid residue having the structure of Formula K-1, and Blood tank having a Raleigh new analogues of amino acid residues, or are independently selected from an isomer thereof;

Figure pat00063
, here

R 3, R 5 and each R 4 is H, -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

R 6 is H or C 1 alkyl;

A is C 3 -C 8 cycloalkyl, C 3 -C 8 heterocycloalkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, 9-10 membered fused bicyclic ring or 13- a 14-membered fused tricyclic ring, where A is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;

L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene, polyalkylene glycols, poly (ethylene glycol), -O (CR 11 R 12 ) k -, -S (CR 11 R 12) k - , -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C (O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11 -, -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k- , -C (O) NR 11 (CR 11 R 12 ) k- , -NR 11 C (O) (CR 11 R 12 ) k- , wherein each of R 11 and R 12 is independently H, C 1 and 8, and the alkyl, k is an integer from 1 to 12, - 8 alkyl, halo-substituted -C 1 - 8 alkyl or hydroxy-substituted -C 1

X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 is selected from 8-alkyl, protecting group, or the terminal functional group Lim), - CH 2 O) p CH 2 CH 2 -X 2 , and any combination of these (where, p is 1 to 10,000, X 2 is H, C 1

At least one AA is a pyrrolysine analog amino acid residue having the structure of Formula A-2 or Formula B-2, or at least one BB is Formula C-1 or Formula D-1 or Formula E-1 or Formula F-1 Or a pyrrolysine analog amino acid residue having the structure of Formula G-1 or Formula H-1 or Formula I-1 or Formula J-1 or Formula K-1 or Formula L-1, or an isomer thereof.

In certain embodiments k is an integer from 1 to 11. In certain embodiments k is an integer from 1 to 10. In certain embodiments k is an integer from 1 to 9. In certain embodiments k is an integer from 1 to 8. In certain embodiments k is an integer from 1 to 7. In certain embodiments k is an integer from 1 to 6. In certain embodiments k is an integer from 1 to 5. In certain embodiments k is an integer from 1 to 4. In certain embodiments k is an integer from 1 to 3. In certain embodiments k is an integer from 1 to 2.

In certain embodiments p is an integer from 1 to 8000. In certain embodiments p is an integer from 1 to 7000. In certain embodiments p is an integer from 1 to 6000. In certain embodiments p is an integer from 1 to 5000. In certain embodiments p is an integer from 1 to 4000. In certain embodiments p is an integer from 1 to 3000. In certain embodiments p is an integer from 1 to 2000. In certain embodiments p is an integer from 1 to 1000. In certain embodiments p is an integer from 1 to 500. In certain embodiments p is an integer from 1 to 400. In certain embodiments p is an integer from 1 to 300. In certain embodiments p is an integer from 1 to 200. In certain embodiments p is an integer from 1 to 100. In certain embodiments p is an integer from 1 to 90. In certain embodiments p is an integer from 1 to 80. In certain embodiments p is an integer from 1 to 70. In certain embodiments p is an integer from 1 to 60. In certain embodiments p is an integer from 1 to 50. In certain embodiments p is an integer from 1 to 40. In certain embodiments p is an integer from 1 to 30. In certain embodiments p is an integer from 1 to 20. In certain embodiments p is an integer from 1 to 10. In certain embodiments p is an integer from 1 to 5.

In certain embodiments n is an integer from 1 to 4000. In certain embodiments n is an integer from 1 to 3000. In certain embodiments n is an integer from 1 to 2000. In certain embodiments n is an integer from 1 to 1000. In certain embodiments n is an integer from 1 to 700. In certain embodiments n is an integer from 1 to 800. In certain embodiments n is an integer from 1 to 600. In certain embodiments n is an integer from 1 to 500. In certain embodiments n is an integer from 1 to 400. In certain embodiments n is an integer from 1 to 300. In certain embodiments n is an integer from 1 to 200. In certain embodiments n is an integer from 1 to 100. In certain embodiments n is an integer from 1 to 90. In certain embodiments n is an integer from 1 to 80. In certain embodiments n is an integer from 1 to 70. In certain embodiments n is an integer from 1 to 60. In certain embodiments n is an integer from 1 to 50. In certain embodiments n is an integer from 1 to 40. In certain embodiments n is an integer from 1 to 30. In certain embodiments n is an integer from 1 to 20. In certain embodiments n is an integer from 1 to 10. In certain embodiments n is an integer from 1 to 5.

In certain embodiments, compounds of Formula III used in the derivatization methods provided herein include, but are not limited to, amino sugars. In certain embodiments, such amino sugars include, but are not limited to, D-mannosamine and D-galactosamine.

In certain embodiments, the compounds of formula IV used in the derivatization methods provided herein include 2-amino-benzaldehyde (2-ABA), derivatives of 2-amino-benzaldehyde (2-ABA), 2- provided herein Derivatives of amino-benzaldehyde (2-ABA), 2-amino-acetophenones (2-AAP), derivatives of 2-amino-acetophenones (2-AAP), 2-amino-acetophenones provided herein Derivatives of AAP), 2-amino-5-nitro-benzophenone (2-ANBP), derivatives of 2-amino-5-nitro-benzophenone (2-ANBP) and 2-amino-5-nitro provided herein Derivatives of -benzophenone (2-ANBP), including but not limited to.

FIG. 22 shows a scheme for the chemical derivatization of PCL-A with 2-amino-benzaldehyde (2-ABA), wherein one or more PCL-As are site-specifically incorporated into the protein. The cyclization reaction of semialdehyde with 2-ABA forms a quinazoline-type moiety (22-A or 22-B) as a Friedlaender-type reaction, which is further reduced with the use of a suitable reducing agent to form tetrahydro Quinazoline type residues (22-D) or substituted anilines (22-E). Alternatively, further reaction of the quinazoline-type residues 22-B forms a fused ring residue (22-C). Suitable reducing agents for reducing the quinazoline-type moiety include, but are not limited to, sodium cyanoborohydride and sodium borohydride.

In addition, FIG. 23 shows protein conjugates of various structures formed after the reaction of PCL-A with 2-ABA, 2-AAP or 2-ANBP. The expected mass increase due to the attachment of these groups was also shown. The structure obtained using this group was characterized by NMR spectroscopy (see Example 45). In addition, NaCNBH 3 reduction has been found to stabilize PCL-based protein conjugates and prevent dissociation of PCL-ABA and PCL-AAP linkages even at high temperatures (see Example 44).

2-ABA and Δ 1 -pyrroline-5-carboxylic acid (L-1-pyrroline-5-carboxylic acid) (Strecker, HJ, (1971), Methods in Enzymology 27B, 254-257), Vogel, HJ, and Davis, BD (1953), "Glutamic gamma-semialdehyde and delta1-pyrroline-5-carboxylic acid, intermediates in the biosynthesis of proline," J. Am. Chem. Soc. 74, 109-112] And Schepf, C., and Oechler, F., (1936), Ann. Chem. 523, 1) and other pyrrolines (Schoepf, C., and Oechler, F., (1936), Ann. ... Chem 523, 1] and [Schoepf, C., Steuer and, H., (1947), Ann Chem 558, 124] view) of the reaction is Δ 1 in proline metabolism and biosynthesis-pyrroline-5-carboxylic It has been used as a colorimetric assay in studying the role of acids (Vogel, HJ, and Davis, BD (1953), "Glutamic gamma-semialdehyde and Δ 1 -pyrroline-5-carboxylic acid, intermediates in the biosynthesis of proline, "J. Am. Chem. Soc. 74, 109-112," Strecker, HJ, (1960), "The interconversion of glutamic acid and proline," J. Biol. Chem. 235, 204 5-2050, Mezl, VA, and Knox, WE, (1976), "Properties and analysis of a stable derivative of pyrroline-5-carboxylic acid for use in metabolic studies," Analytical Biochemistry 74, 430-40, Wu, GY, and Seifter, S., (1975), "A new method for the preparation of delta 1-pyrroline 5-carboxylic acid and proline," Analytical Biochemistry 67, 413-21, Williams, I., and Frank, L., (1975), "Improved chemical synthesis and enzymatic assay of Δ 1 -pyrroline-5-carboxylic acid," Anal. Biochem. 64, 85-97 and Strecker, HJ, (1957), "The interconversion of glutamic acid and proline," J. Biol. Chem. 225, 825). The scheme shows the formation of glutamic acid-gamma-semialdehyde intermediates, but the reaction can proceed directly from pyrroline-carboxy-lysine without the formation of such intermediates.

High resolution mass spectrometric studies of model protein hRBP4 demonstrated chemical derivatization of PCL residues by 2-ABA, where PCL was site specificly incorporated into protein hRBP4 (see FIGS. 25-26, Example 11). 24 shows a mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-ABA, wherein the protein derivatized with 2-ABA produced a major peak at 23269.2 Da, with the least amount of unmodified protein at 23166.8 Da Detected. The mass increase of 102.4 Da for the derivatized protein is consistent with the expected increase of 103 Da for the attachment of 2-ABA residues, as shown in FIG. 23, so that proteins with site specific incorporation of PCL are site specific at the PCL site. Proves to be modified as MS / MS analysis (FIG. 25) obtained after LC-MS analysis of trypsin digest of 2-ABA-derivatized hRBP4 Phe122PCL protein confirmed the expected YWGVASF * LQK peptide, where F * was 2-ABA- Have a mass consistent with the strain PCL. 25A is a fragmentation pattern of the YWGVASF * LQK peptide, where F * has a mass consistent with 2-ABA-modified PCL. 25B is the TIC (total ion chromatogram) and EIC (extract ion chromatogram) of 2+ ions of YWGVASF * LQK (F * = PCL and PCL-2-ABA adducts), wherein the derivatized species and derivatization Comparison of EIC to non-detectable species indicates completion of reaction. FIG. 25C is a mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-ABA, with 3+ and 2+ precursors of YWGVASF * LQK (F * = PCL-2-ABA adduct) m / z 459.92 (3+) And 689.37 (2+), respectively, demonstrate that the reaction observed when using 2-ABA occurs site-specific with the PCL residue incorporated at the desired TAG site of residue 122.

Evaluation of the pH dependence of derivatization is shown in FIG. 26. Mass spectra of hRBP4 (hRBP4 Phe122PCL) with PCL incorporated at position 122 are shown in FIG. 26A, where hRBP4 Phe122PCL did not react with 2-ABA, and FIGS. 26B and 26C showed hRBP4 Phe122PCL to pH 5.0 Mass spectrum after reaction with 10 mM 2-ABA in buffer and 10 mM 2-ABA in phosphate buffer, pH 7.4, respectively. The reaction of the PCL residue of the hRBP4 protein with 2-ABA proceeds rapidly up to 95% complete at room temperature in aqueous buffer adjusted to pH 5 and to approximately 87% to slightly lower at pH 7.4. The reaction with 2-ABA is selective for PCL residues as shown in FIGS. 27D-F, wherein hRBP4 labeled with inert non-natural amino acid O-methyl-phenylalanine (OMePhe) at position 62 and wild type phenylalanine (Phe) at position 122 No reaction with 2-ABA was observed for hRBP4 with) residues.

An evaluation of the reaction efficiency as a function of the reactant: protein concentration ratio and the reactivity with the 2-ABA-like reactant is shown in FIG. 27. Mass spectra after reaction of hRBP4 (hRBP4 Phe122PCL) with 0.1 mM 2-amino-benzaldehyde (2-ABA) with PCL incorporated at position 122 are shown in FIG. 27A, and 0.1 mM 2-amino-acetophenone of hRBP4 Phe122PCL ( 2-AAP) and mass spectra after reaction with 0.1 mM 2-amino-5-nitro-benzophenone (2-ANBP) are shown in FIGS. 27B and 27C, respectively. All reactions were performed in 200 mM sodium acetate, pH 5.0. As in FIG. 23, the protein conjugate was detected to have the expected mass increase at the correct mass. In addition, yields of greater than 88% conversion were achieved for 2-ABA and 2-AAP, but about 5% yield was achieved for 2-ANBP due to low solubility. However, this demonstrates that the 2-ABA analog reacts efficiently at the PCL incorporation site when the reactant: protein concentration ratio is 6: 1. The degree of reaction was only slightly lower than when the reactant: protein ratio was performed at 600: 1 (FIG. 26).

There was no significant improvement in the degree of derivatization when the derivatizing agent was added in the final concentration range of 0.1-10 mM corresponding to a 6 to 600-fold molar excess of protein. In addition, at molar ratios above 4700 additional protein residues expected to be lysine were derivatized with 2-ABA (FIG. 28). In FIG. 28, significant excess of 2-ABA relative to protein in 10 × PBS was determined by PCL incorporated hRBP4 (A, about 17 μM, 4700 fold excess of 2-ABA relative to protein) and OMePhe incorporated hRBP4 (B, about 6.5 μM, 15400-fold excess) led to conjugation at additional sites, illustrating that conjugation at these additional sites is mediated by residues other than PCL.

To further illustrate the usefulness of the methods provided herein, FIG. 29 shows before and after derivatization of PCL incorporated into FAS-TE with 2-amino-acetophenone (2-AAP) at pH 5.0 and pH 7.4. Mass spectra are shown (see Example 12). 29A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL and FIG. 29B shows the mass spectrum of the reaction mixture at pH 5.0. Here, 100% of the observable peak intensity occurs at 33318.8 Da, which is 116.8 Da larger than for the unreacted material, as a mass increase of 117 Da is expected for the 2-AAP modified FAS-TE Tyr2454PCL. Similarly, at pH 7.4 the reaction proceeded to 95% completion (FIG. 29C (unreacted) and FIG. 29D (reacted)).

30 is a general scheme for site-specific modification of proteins via chemical derivatization of pyrrolysine and / or PCL using 2-amino-benzaldehyde or 2-amino-benzaldehyde analogs. Certain embodiments of such analogs are provided herein. The reaction of 2-ABA with the pyrroline ring of pyrrolysine and PCL is similar to that of 2-ABA with Δ 1 -pyrroline-5-carboxylic acid. Similarly, the reaction of pyrrolysine and PCL with 2-AAP and 2-ANBP yields a protein modified with each substituted residue in FIG. 23.

In certain embodiments, 2-ABA functionality is used to site-specifically attach various groups to proteins, polypeptides and / or peptides having pyrrolysine or PCL incorporated therein. Such groups include labels, dyes, polymers, water soluble polymers, polyalkylene glycols, poly (ethylene glycol), derivatives of poly (ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X 2, and any combination thereof (wherein, p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl include, but, the protecting group or the terminal functional group Im) However, the present invention is not limited thereto.

In certain embodiments, 2-AAP functionality is used to site-specifically attach various groups to proteins, polypeptides and / or peptides having pyrrolysine or PCL incorporated therein. Such groups include labels, dyes, polymers, water soluble polymers, polyalkylene glycols, poly (ethylene glycol), derivatives of poly (ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X 2, and any combination thereof (wherein, p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl include, but, the protecting group or the terminal functional group Im) However, the present invention is not limited thereto.

In certain embodiments, 2-ANPA functionality is used to site-specifically attach various groups to proteins, polypeptides and / or peptides having pyrrolysine or PCL incorporated therein. Such groups include labels, dyes, polymers, water soluble polymers, polyalkylene glycols, poly (ethylene glycol), derivatives of poly (ethylene glycol), sugars, lipids, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X 2, and any combination thereof (wherein, p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl include, but, the protecting group or the terminal functional group Im) However, the present invention is not limited thereto.

Any of the above mentioned groups are attached to 2-ABA, 2-AAP and 2-ANPA using any of the attachment points R 7 to R 11 shown in FIG. 30. In other embodiments, the benzene ring is replaced with any other ring structure provided herein, including but not limited to naphthalene. In other embodiments, the benzene ring is replaced with a sugar.

The concentration of the derivatizing agent used in the methods provided herein for derivatization of pyrrolysine and PCL incorporated into proteins, polypeptides and / or peptides ranges from about 0.005 mM to about 50 mM. In certain embodiments, the concentration of derivatization agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 25 mM. In certain embodiments, the concentration of derivatization agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 10 mM. In certain embodiments, the concentration of derivatization agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 5 mM. In certain embodiments, the concentration of derivatization agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 2 mM. In certain embodiments, the concentration of derivatization agent used in the methods provided herein for derivatization of pyrrolysine or PCL ranges from about 0.005 mM to about 1 mM.

In the derivatization methods provided herein, the molar ratio of the derivatization agent to a protein, polypeptide or peptide having one or more pyrrolysine or PCL incorporated therein is in the range of about 0.05 to about 10000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 8000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 6000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 4000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 2000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 1000. In certain embodiments, the molar ratio of the derivatizing agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 800. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 600. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 400. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 200. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 100. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 50. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 25. In certain embodiments, the molar ratio of the derivatizing agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 10. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 0.05 to about 1.

In other embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 10000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 8000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 6000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 4000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 2000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 1000. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 800. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 600. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 400. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 200. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 100. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 50. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 25. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 1.5 to about 10. In certain embodiments, the molar ratio of the derivatization agent to such proteins, polypeptides or peptides ranges from about 6 to about 600.

The pH of the buffer solution used for derivatization of proteins, polypeptides and / or peptides using the methods and compositions provided herein is from about pH 2 to about pH 10. In certain embodiments, the pH is from about pH 4 to about pH 8. In certain embodiments, the pH is from about pH 4 to about pH 7.5. In certain embodiments, the pH is about pH 4 to about pH 7. In certain embodiments, the pH is from about pH 4 to about pH 6.5. In certain embodiments, the pH is from about pH 4 to about pH 6. In certain embodiments, the pH is from about pH 4 to about pH 5.5. In certain embodiments, the pH is from about pH 4 to about pH 5. In certain embodiments, the pH is from about pH 4 to about pH 4.5. In certain embodiments, the pH is from about pH 6 to about pH 8. In certain embodiments, the pH is from about pH 6 to about pH 7.5. In certain embodiments, the pH is from about pH 6 to about pH 7. In certain embodiments, the pH is from about pH 6 to about pH 6.5. In certain embodiments, the pH is from about pH 7 to about pH 8. In certain embodiments, the pH is from about pH 7 to about pH 7.5.

The derivatization methods provided herein are useful for derivatization of pyrrolysine or PCL that is site-specifically incorporated into a protein. In certain embodiments, such derivatization methods are used to derivatize a PCL residue incorporated at a single site into a protein, polypeptide and / or peptide. In other embodiments, such derivatization methods are used to derivatize PCL residues incorporated at multiple sites in proteins, polypeptides and / or peptides. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, derivatized proteins, polypeptides or peptides using the methods and compositions provided herein , 10, 11, 12, 13, 14, 15 or more pyrrolysine or pyrrolysine analogs.

In another aspect provided herein, proteins, polypeptides and peptides having one or more pyrrolysine or PCL incorporated therein are derivatized by at least one simultaneous or post-translational modification. Non-limiting examples of such translational co- or post-translational modifications include glycosylation, acetylation, acylation, methylation, nitration, sulfated, lipid-modified, palmitoylated, palmitate addition, phosphorylation, glycolipid-linked modifications, and the like. Included, but not limited to. Also included in this aspect are methods of producing, purifying, characterizing and using such proteins, polypeptides and peptides containing at least one such translational simultaneous or post-translational modification.

Biotherapeutics with Site-Specific Modifications

Coupled to PCL and pyrrolysine incorporated into proteins, polypeptides and / or peptides Macromolecular polymer

In certain embodiments, macromolecular polymers are added to pyrrolysine or PCL residues incorporated into proteins, polypeptides and / or peptides using the compositions, methods, techniques and strategies described herein. A wide variety of macromolecular polymers can be coupled to pyrrolysine and PCL residues incorporated into the proteins, polypeptides and / or peptides described herein. Such modifications are used to adjust the biological properties of such proteins, polypeptides and / or peptides and / or to provide new biological properties to these proteins, polypeptides and / or peptides. In certain embodiments, the macromolecular polymers are coupled to such proteins, polypeptides and / or peptides through direct coupling to pyrrolysine or PCL residue (s) incorporated into the proteins, polypeptides and / or peptides provided herein. . In other embodiments, the macromolecular polymers are coupled to proteins, polypeptides and / or peptides provided herein via bi-, tri-, tetra-, and multi-functional linkers coupled to pyrrolysine or PCL residue (s). do. In other embodiments, the macromolecular polymer is coupled to a protein, polypeptide and / or peptide provided herein via a bifunctional linker coupled to pyrrolysine or PCL residue (s). In certain embodiments, such di-, tri-, tetra-, and multi-functional linkers are monofunctional linkers whose ends are substituents specific for reaction with pyrrolysine or PCL residues. In certain embodiments, such di-, tri-, tetra-, and multi-functional linkers are substituents whose one or more terminus is specific for reaction with pyrrolysine or PCL residue (s) and the other terminus is pyrrolysine or PCL Another functional substituent that does not react with the residue (s) is a heterofunctional linker. Specific substituents that are pyrrolysine or PCL specific reactive groups are provided herein and other functional substituents and resulting linkages used in such di-, tri-, tetra-, and multi-functional linkers include, but are not limited to, those listed in Table 1 It is not limited.

Figure pat00064

Covalent attachment of macromolecular polymers to biologically active molecules such as proteins, polypeptides and / or peptides provided herein can result in an increase in water solubility (eg, water solubility in a physiological environment), an increase in bioavailability, serum half-life Approaches to increase, increase therapeutic half-life, adjustment of immunogenicity, adjustment of biological activity, or extension of circulation time of such biologically active molecules. Important features of such macromolecular polymers include the absence of biocompatibility, nontoxicity, and immunogenicity, and the treatment of proteins, polypeptides and / or peptides provided herein coupled to macromolecular polymers via pyrrolysine or PCL residues. Such macromolecular polymers are pharmaceutically acceptable for use.

Certain macromolecular polymers coupled to pyrrolysine or PCL residue (s) incorporated into the proteins, polypeptides and / or peptides described herein are water soluble polymers. In certain embodiments such water soluble polymers are coupled via pyrrolysine or PCL residue (s) incorporated into proteins, polypeptides and / or peptides provided herein, and in other embodiments such water soluble polymers are provided herein. To any of these proteins, polypeptides and / or peptides via any functional or substituent group coupled to the pyrrolysine or PCL residue (s) incorporated into the polypeptide and / or peptide. In some embodiments, proteins, polypeptides and / or peptides provided herein comprise one or more PCL residues coupled to a water soluble polymer and one or more naturally occurring amino acids linked to a water soluble polymer.

Structural forms of macromolecular polymers coupled to pyrrolysine or PCL residues incorporated into proteins, polypeptides and / or peptides include, but are not limited to, linear, forked or branched polymers. In certain embodiments, the backbone of such branched or forked water soluble polymers has 2 to about 300 ends. In certain embodiments, each end of such a multifunctional polymer derivative, for example, but not limited to, a linear polymer having two ends, comprises a functional group. In certain embodiments such functional groups are the same and in other embodiments such functional groups are different. Non-limiting examples of such terminal functional groups include N-succinimidyl carbonate, amine, hydrazide, succinimidyl propionate and succinimidyl butanoate, succinimidyl succinate, succinimidyl ester, benzotria Sol carbonates, glycidyl ethers, oxycarbonylimidazoles, p-nitrophenyl carbonates, aldehydes, maleimides, ortopyridyl-disulfides, acrylols and vinylsulfones, including but not limited to Do not. In other embodiments, functional groups include those listed in Table 1.

Covalent attachment of water soluble polymers (also referred to herein as hydrophilic polymers) to biologically active molecules such as proteins, polypeptides and / or peptides provided herein is water soluble (eg, water soluble in a physiological environment). Approaches to increase of, increase of bioavailability, increase of serum half-life, increase of therapeutic half-life, adjustment of immunogenicity, adjustment of biological activity, or extension of circulation time of such biologically active molecules. Important features of such water soluble polymers include the absence of biocompatibility, nontoxicity, and immunogenicity, and for the therapeutic use of proteins, polypeptides and / or peptides provided herein coupled to water soluble polymers via pyrrolysine or PCL residues. Such macromolecular polymers are pharmaceutically acceptable.

Hydrophilic polymers coupled to proteins, polypeptides and / or peptides via pyrrolysine or PCL residues include polyalkyl ethers and alkoxy-capped analogs thereof, polyvinylpyrrolidone, polyvinylalkyl ethers, polyoxazolines, polyalkyls Oxazoline, polyhydroxyalkyl oxazoline, polyacrylamide, polyalkyl acrylamide, polyhydroxyalkyl acrylamide, polyhydroxyalkyl acrylate, polysialic acid and analogs thereof, hydrophilic peptide sequences; Polysaccharides and derivatives thereof, cellulose and derivatives thereof, chitin and derivatives thereof, hyaluronic acid and derivatives thereof, starch, alginate, chondroitin sulfate, albumin, pullulan, carboxymethyl pullulan, polyamino acids and derivatives thereof, maleic acid Anhydride copolymers, polyvinyl alcohol and copolymers thereof, polyvinyl alcohol and terpolymers thereof, and combinations thereof, including but not limited to.

Such polyalkyl ethers and alkoxy-capped analogs thereof include polyoxyethylene glycol (also known as poly (ethylene glycol) or PEG), polyoxyethylene / propylene glycol, and methoxy or ethoxy-capped analogs thereof. Include, but are not limited to. Such polyhydroxyalkyl acrylamides include, but are not limited to, polyhydroxypropylmethacrylamide and derivatives thereof. Such polysaccharides and derivatives thereof include, but are not limited to, dextran and dextran derivatives (eg (only as examples), carboxymethyldextran, dextran sulfate and aminodextran). Such cellulose and its derivatives include, but are not limited to, carboxymethyl cellulose and hydroxyalkyl cellulose. Such chitin and its derivatives include, but are not limited to, chitosan, succinyl chitosan, carboxymethyl chitin and carboxymethyl chitosan. Such polyamino acids and derivatives thereof include, but are not limited to, polyglutamic acid, polylysine, polyaspartic acid and polyaspartamide. Such maleic anhydride copolymers include, but are not limited to, styrene maleic anhydride copolymers and divinylethyl ether maleic anhydride copolymers.

In some embodiments, a water soluble polymer coupled directly or indirectly to such protein, polypeptide and / or peptide via a pyrrolysine or PCL residue (s) incorporated into a protein, polypeptide and / or peptide provided herein is a poly (Ethylene glycol) (PEG). Poly (ethylene glycol) is considered biocompatible, and PEG can coexist with it without causing harm to living tissues or organisms. PEG is also substantially non-immunogenic and therefore does not tend to elicit an immune response in the body. PEG is a hydrophilic polymer that has been widely used in pharmaceuticals, artificial implants, and other applications where biocompatibility, nontoxicity, and the absence of immunogenicity are important. PEG conjugates tend not to cause a substantial immune response or to cause blood clotting or other undesirable effects. Thus, when attached to a molecule having some desirable function in the body, such as a biologically active agent, PEG tends to mask the agent and can reduce or eliminate any immune response such that the organism will allow for the presence of that agent. To be able.

In some embodiments, poly (ethylene glycol) coupled directly or indirectly to such proteins, polypeptides and / or peptides via pyrrolysine or PCL residue (s) incorporated into the proteins, polypeptides and / or peptides provided herein This is a straight chain polymer and in other embodiments such PEG polymer is a branched polymer. The molecular weight or molecular weight distribution of such straight and branched PEG polymers is from about 100 Da to about 100,000 Da or higher. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is between about 100 Da and 50,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is about 100 Da to 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is about 1,000 Da to 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is about 5,000 Da to 40,000 Da. In some embodiments, the molecular weight or molecular weight distribution of such PEG polymers is about 10,000 Da to 40,000 Da. In certain embodiments, the molecular weight of such PEG polymers is 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da , 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da or 100 Da.

Structural forms of PEG polymers coupled to pyrrolysine or PCL residue (s) incorporated into proteins, polypeptides and / or peptides include, but are not limited to, linear, forked or branched. In certain embodiments, the backbone of such branched or forked PEG polymers has 2 to about 300 ends. In certain embodiments, each end of such a multifunctional polymer derivative, for example, but not limited to, a linear polymer having two ends, comprises a functional group. In certain embodiments such functional groups are identical and in other embodiments at least one of these functional groups is different. In other embodiments, these functional groups are different.

In certain embodiments, at least one terminal of the PEG polymer comprises a functional group prior to coupling with the protein, polypeptide and / or peptide. In certain embodiments PEG is a linear polymer having functional groups at one end, and in other embodiments PEG is a linear polymer having functional groups at each end to form a bifunctional PEG polymer. In other embodiments PEG is a fork polymer having functional groups at one end, and in other embodiments PEG is a fork polymer having functional groups at two or more ends. In other embodiments, the PEG is a fork polymer having functional groups at each end to form a multifunctional PEG polymer. In other embodiments, PEG is a branched polymer having functional groups at one end, and in other embodiments, PEG is a branched polymer having functional groups at two or more ends. In other embodiments, the PEG is a branched polymer with functional groups at each end to form a multifunctional PEG polymer. In certain embodiments of such aforementioned PEG polymers the functional groups are the same, and in other embodiments of such functional groups at least one functional group is different. In other embodiments of such aforementioned PEG polymers, the functional groups are different. However, at least one terminus of the PEG polymer is available for reaction with at least one pyrrolysine or PCL residue incorporated into the protein, polypeptide and / or peptide.

Non-limiting examples of terminal functional groups include N-succinimidyl carbonate, amine, hydrazide, succinimidyl propionate, succinimidyl butanoate, succinimidyl succinate, succinimidyl ester, benzotriazole Carbonates, glycidyl ethers, oxycarbonylimidazoles, p-nitrophenyl carbonates, aldehydes, maleimides, orthopyridyl-disulfides, acrylols, vinylsulfones, activated carbonates (eg For example, but not limited to, p-nitrophenyl esters, activated esters (for example, but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester), oxime, carbonyl , Dicarbonyl, hydroxylamine, hydroxyl, methoxy, benzaldehyde, acetophenone, 2-amino-benzaldehyde, 2-amino-acetophenone and 2-amino-5-nitro-benzophenone Does not.

FIG. 31 shows one embodiment of the functionalized PEG polymer coupled to the protein via at least one PCL residue incorporated into the protein, 2-amino-acetophenone-PEG 8 (2-AAP-PEG8; TU3205-044) To illustrate. As shown, the 2-amino-acetophenone residue of 2-AAP-PEG8 forms a quinazoline-type residue as a spacer between PEG 8 and the protein. Such quinazoline-type residues may be further reacted to form a fused ring structure. For coupling of 2-AAP-PEG8, this derivatization adds 556 Da to the mass of the protein. Other functionalized PEGs used in the methods provided herein include, but are not limited to, those described in Example 20.

FIG. 32 shows mass spectra of hRBP4 (hRBP4 Phe122PCL) with PCL incorporated at position 122 before and after derivatization with 2-AAP-PEG8 at pH 7.5 (FIG. 32A) and pH 5.0 (FIG. 32B). . This coupling reaction was performed at pH 7.5 and pH 5, where 89% to 100% conversion occurred, with an expected mass increase of 556 Da relative to unreacted protein (FIG. 32C). Wild type hRBP4 did not react with 450 or 2300 fold excess of 2-AAP-PEG8 (FIGS. 32D-F). Since no coupling occurs in the absence of PCL, this further illustrates the specificity of the coupling reaction between PCL and 2AAP-PEG8.

To further illustrate the utility of this labeling method, the thioesterase domain of human fatty acid synthetase (FAS-TE) (FAS-TE Tyr2454PCL) with PCL incorporated at position 2454, generated in Escherichia coli, Derivatized with 2-AAP-PEG8 (see FIG. 33, Example 14). FIG. 33A shows the mass spectrum of unreacted protein (mass 33202 Da), and FIG. 33B shows the mass spectrum of FAS-TE Tyr2454PCL with expected mass 33756 Da by reaction with 2-AAP-PEG8 (TU3205-044) until completion. Shows. Further examples are provided in FIGS. 33C and 33D, which show FAS-TE (FAS-TE Tyr2454PCL) with PCL incorporated at position 2454, produced in Escherichia coli, at room temperature (FIG. 33C) and at 4 ° C. (FIG. 33D) Mass spectrum after derivatization with 2.4 kDa 2-AAP-PEG (TU3205-048). Under the conditions used, only approximately 25% conversion was achieved to yield a protein of expected mass 35585 Da.

34 also shows PEGylation of FAS-TE Tyr2454PCL using 2.4 kDa 2-AAP-PEG and 23 kDa 2-AAP-PEG in the molar ratios shown in the figures. The 0.5 kDa 2-AAP-PEG (2-AAP-PEG8) PEGylation product was not isolated on SDS-PAGE but was verified by mass spectrometry.

The methods provided herein were used to incorporate PCL at 20 positions into fibroblast growth factor 21 (FGF-21) followed by PEGylation of the corresponding PCL residues (see Example 15). 35 shows mass spectra obtained before and after derivatization of FGF21 Lys84PCL with 2-AAP-PEG8. 35A is the mass spectrum of unreacted FGF21 Lys84PCL (21235.6 Da), and FIG. 35B is the mass spectrum of FGF21 Lys84PCL reacted with 2-AAP-PEG8. The PEGylation reaction proceeded to completion and produced a protein of 21792.4 Da. The increase of 556.8 Da is consistent with the expected 556 Da increase for the derivatization. 36 shows the SDS-PAGE results obtained after derivatization of seven FGF21 PCL mutants with 23 kDa 2-AAP-PEG. 36A is an SDS gel of FGF21 PCL mutant (0.1-0.4 mM) reaction mixture reacted with 23 kDa 2-AAP-PEG (pH 7.4, 4 ° C., 60 hours), PEG-FGF21, full length (FL) FGF21- PCL and truncated (TR) FGF21-PCL are shown. 36B shows SDS-PAGE results of eight FGF21 PCL mutants after partial purification of PEG-FGF21, full length (FL) and truncated (TR) FGF21.

The methods provided herein were used to incorporate PCL at 11 positions into mouse erythropoietin (EPO) and then PEGylate the corresponding PCL residues for the three EPO PCL mutants (see Example 6). 37 is an SDS gel obtained after PEGylation of mouse EPO PCL mutant. Mouse EPO with PCL incorporated at three different positions in HEK293F cells was reacted with 2-AAP-mPEG23k (TU3205-052). PEGylated and un PEGylated EPO are indicated by arrows.

In certain embodiments, the bifunctional or multifunctional PEG polymer linked to the protein, polypeptide and / or peptide via PCL residue (s) is further derivatized or substituted with the remaining functional groups on the unreacted terminus. In certain embodiments, bifunctional or polyfunctional PEG polymers linked to proteins, polypeptides and / or peptides via reaction of benzaldehyde, benzophenone or acetophenone residues with PCL residue (s) are added using the remaining functional groups on the unreacted terminus. Derivatization or substitution with

The methods and compositions described herein provide highly efficient methods for the selective modification of proteins, polypeptides and peptides using PEG derivatives, which suitably after the incorporation of the amino acid PCL or pyrrolysine into the protein in response to the selector codon Modifying the corresponding amino acid residues using functionalized PEG derivatives. Accordingly, provided herein are proteins, polypeptides and / or peptides having PCL or pyrrolysine residue (s) incorporated therein, and also herein are water soluble polymers, such as poly, via such PCL or pyrrolysine residue (s). Provided are such proteins, polypeptides and / or peptides linked to (ethylene glycol) (PEG).

The degree of PEGylation of the proteins, polypeptides and / or peptides described herein (ie, the number of PEG polymers linked to the proteins, polypeptides and / or peptides) are adjustable to provide altered pharmacological, pharmacokinetic or pharmacodynamic features. In certain embodiments this change is an increase in this feature, and in other embodiments such a change is a decrease in this feature. In certain embodiments, this feature is an in vivo half life. In some embodiments, the half-life of PEGylated proteins, polypeptides and / or peptides using the methods and compositions provided herein is at least about 10%, 20%, 30%, 40 compared to unmodified proteins, polypeptides and / or peptides. %, 50%, 60%, 70%, 80%, 90%, 2 times, 5 times, 10 times, 50 times, 100 times or at least about 200 times increased.

In certain embodiments, proteins, polypeptides, and / or peptides PEGylated using the methods and compositions provided herein include hydrophobic chromatography, affinity chromatography; Anion exchange chromatography, cation exchange chromatography, Q quaternary ammonium resin, DEAE Sepharose, chromatography on silica; Reverse phase HPLC, gel filtration (eg, but not limited to, SEPHADEX G-75, Sephacryl S-100, SUPERDEX 75, 100 and 200), hydrophobic interaction Functional chromatography, size-exclusion chromatography, metal-chelate chromatography; Ultrafiltration / diafiltration, ethanol precipitation, chromatographic focusing, alternative chromatography, electrophoretic procedures (e.g., but not limited to, prepotential focusing for purification), differential solubility (e.g., but not limited to ), And ammonium sulfate precipitation) and extraction.

In another aspect, PEG polymers used in the methods provided herein have weak or degradable linkages in the backbone. By way of example only, such PEG polymers have ester linkages that are subject to hydrolysis in the polymer backbone, and such hydrolysis cleaves the linkages to release proteins, polypeptides or peptides to which PEG is attached.

In another aspect, the methods and compositions described herein are used to create a substantially homogeneous formulation of a polymer-protein conjugate. As used herein, "substantially homogeneous" means that the polymer: protein conjugate molecule is observed to be more than half of the total protein. The polymer: protein conjugate has biological activity, and the "substantially homogeneous" PEGylated polypeptide formulations of the invention provided herein provide the benefits of homogeneous formulations, e.g. to lot) is a homogeneous agent that is sufficiently homogeneous to demonstrate its ease of clinical use in pharmacokinetic prediction.

In another aspect, the methods and compositions described herein are used to create a substantially homogeneous formulation of a polymer: protein conjugate. As used herein, "substantially homogeneous" means that the polymer: protein conjugate molecule is observed to be more than half of the total protein. Polymer: protein conjugates have biological activity, and the "substantially homogeneous" PEGylated polypeptide formulations of the invention provided herein provide the benefits of homogeneous formulations, e.g. pharmacokinetic prediction per product number, by way of example only. It is a formulation that is sufficiently homogeneous to show its ease of clinical use.

In another aspect, using the methods and compositions described herein to prepare a mixture of polymer-protein conjugate molecules, an advantage provided herein is that the ratio of mono-polymer: protein conjugate to include in the mixture is selectable. Thus, in certain embodiments, mixtures of various proteins to which various numbers of polymeric moieties (ie, non-, tri-, tetra-, etc.) are attached are prepared, and these conjugates are mono-polymers-prepared by the methods described herein. Optional combination with a protein conjugate produces a mixture with a proportion of mono-polymer: protein conjugate.

The ratio of poly (ethylene glycol) molecules to protein molecules will depend on their concentration in the reaction mixture. In certain embodiments, the ratio of optimal (optimal in terms of reaction efficiency with minimal unreacted protein or polymer) is determined by the molecular weight of the selected poly (ethylene glycol) and the number of reactive groups available. The higher the molecular weight of the polymer, the fewer polymer molecules attached to the protein. Similarly, in other embodiments, branching of PEG polymers is considered in the optimization of these parameters. In this example, the higher the molecular weight (or the greater the number of branches), the higher the polymer: protein ratio.

Direct coupling of amino sugars with pyrrolysine and / or PCL incorporated into proteins, polypeptides and / or peptides

In another aspect provided herein, proteins, polypeptides and / or peptides having one or more pyrrolysine and / or PCL residue (s) incorporated therein are derivatized with amino sugars. 38 shows a general scheme for coupling D-mannosamine to a protein with PCL incorporated therein (illustrated as R 1 ). In this embodiment, the aminoaldehyde residue of D-mannosamine reacts with the PCL residue to increase the mass of the protein by 161 Da. Other amino sugars used in this embodiment include, but are not limited to, mannosamine, galactosamine and glucosamine. FIG. 39 shows the mass spectrum after reaction of hRBP4 (hRBP4 Phe122PCL) with mannosamine with PCL incorporated at position 122, FIG. 40 shows human fatty acid synthetase (FAS-TE) with PCL incorporated at position 2222 Mass spectra of (FAS-TE Leu2222PCL / Leu2223Ile) before (Figure 40A) and after (Figure 40B) reaction with mannosamine.

Glycosylation of such proteins, polypeptides and / or peptides via coupling with pyrrolysine and PCL incorporated into proteins, polypeptides and / or peptides

The methods and compositions described herein are used to obtain proteins, polypeptides and peptides having one or more pyrrolysine and / or PCL residues bearing saccharide residues. The saccharide residues may be natural (eg, but not limited to, N-acetylglucosamine and D-mannosamine) or non-natural (eg, but not limited to, 3-fluorogalactose) Can be. In certain embodiments, the saccharide is linked to pyrrolysine and / or PCL residues by non-natural linkage, including but not limited to the formation of quinazoline-type residues. In certain embodiments, the saccharide is linked to pyrrolysine and / or PCL residues by non-natural linkage, including but not limited to the formation of quinazoline-type residues further reduced using a reducing agent. In certain embodiments, the saccharide is pyrrolysine by non-natural linkage, including but not limited to the formation of quinazoline-type residues that further react to form a fused ring system as provided herein. And / or to PCL residues (see FIGS. 22, 23 and 30). In certain embodiments, the addition of saccharide (s), including but not limited to addition of glycosyl residues to proteins, polypeptides and peptides having one or more pyrrolysine and / or PCL incorporated therein, Occurs in vivo. In other embodiments, the addition of saccharide (s), including but not limited to addition of glycosyl residues to proteins, polypeptides and peptides having one or more pyrrolysine and / or PCL incorporated therein, It occurs in vitro. In another embodiment, the saccharide once attached to a pyrrolysine and / or PCL residue is further modified by glycosyltransferase and / or other enzymatic treatment to incorporate one or more pyrrolysine and / or PCL therein. To generate oligosaccharides bound to proteins, polypeptides or peptides having

FIG. 41 illustrates an embodiment wherein oligosaccharides are attached to a protein, polypeptide, or peptide through the formation of a quinazoline-type residue, either concurrent or post-translational modification. In certain embodiments, the saccharide is linked to pyrrolysine and / or PCL residues by non-natural linkage, including but not limited to the formation of quinazoline-type residues further reduced using a reducing agent. In certain embodiments, the saccharide is pyrrolysine by non-natural linkage, including but not limited to the formation of quinazoline-type residues that further react to form a fused ring system as provided herein. And / or PCL residues (FIGS. 22, 23 and 30). In this embodiment, by way of example only, an oligosaccharide is linked to (GlcNAc-) a 2-amino-acetophenone (2-AAP) conjugated to pyrrolysine and / or PCL residues incorporated into a protein, polypeptide or peptide. Man) 2 -Man-GlcNAc-GlcNAc core. In another embodiment, the oligosaccharide comprises a (GlcNAc-Man) 2 -Man-GlcNAc-GlcNAc core linked to 2-amino-benzaldehyde (2-ABA) and incorporated into a protein, polypeptide or peptide with 2-ABA. Protein conjugates are formed by the reaction of pyrrolysine and / or PCL residues. In another embodiment, the oligosaccharide comprises a (GlcNAc-Man) 2 -Man-GlcNAc-GlcNAc core linked to 2-amino-benzophenone (2-ABP) and incorporates 2-ABP with a protein, polypeptide or peptide The protein conjugate is formed by the reaction of pyrrolysine and / or PCL residues. Other oligosaccharides used in this embodiment have a 2-ABA, 2-AAP or 2-ANBP residue.

By the covalent attachment of the protein alignment, heterodimer dimer, trimer yijongsam, two kinds of oligomers, such paper-mer, homologous trimer, oligomer homogeneous, asymmetric homodimer, forming a homogeneous asymmetric trimer and asymmetric homogeneous oligomer

In another aspect using the methods provided herein, heterodimers, heterotrimers, heteromultimers, using site specific incorporation of one or more pyrrolysine and / or PCL into proteins, polypeptides and / or peptides, Forms protein-protein conjugates including, but not limited to, homodimers, homotrimers, homomultimers, asymmetric homodimers, asymmetric homotrimers, and asymmetric homodimers. Such protein-protein conjugate formation is performed using site specific crosslinking between proteins having pyrrolysine and PCL residue (s) incorporated therein. FIG. 42 illustrates certain embodiments of such protein-protein conjugate formation (heterodimers, heterotrimers, homotrimers) in which proteins having PCL residue (s) incorporated therein are crosslinked. In Figure 42 the protein conjugate linkage formed by this crosslinking is a quinazoline-type residue that connects the proteins together, but in other embodiments the linkage is a reduced form of the quinazoline-type residue (FIGS. 22, 23 and 30). In other embodiments, the linking is a fused ring residue (FIGS. 22, 23 and 30).

Non-limiting examples of such protein-protein conjugates include, but are not limited to: protein-protein conjugate cytokines, growth factors, growth factor receptors, interferons, interleukins, inflammatory molecules, oncogene products, peptide hormones, signals Transduction molecules, steroid hormone receptors, transcriptional activators, transcriptional inhibitors, erythropoietin (EPO), fibroblast growth factor, fibroblast growth factor 21 (FGF21), leptin, insulin, human growth hormone, epidermal neutrophil activating peptide-78 , GROα / MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, playotropin, SCF, c-kit ligand, VEGF, G-CSF, IL-1, IL-2, IL-8, IGF-I, IGF-II, FGF (fibroblast growth factor), PDGF, TNF, TGF-α, TGF -ß, EGF (epidermal growth factor), KGF (keratinocyte growth factor), CD40L / CD40, VLA-4 / VCAM-1, I CAM-1 / LFA-1, hyalurin / CD44, Mos, Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and / or corticosteroid. In another set of embodiments, the protein is homologous to the following therapeutic or other proteins: alpha-1 anti-trypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, Atrial natriuretic polypeptide, atrial peptide, CXC chemokine, T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4 , MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein- 1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, C-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine Epithelial neutrophil activating peptide-78, GROα / MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1, Blood growth factor (EGF), epidermal neutrophil activating peptide, exfoliating toxin, factor IX, factor VII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebro Sidase, gonadotropin, growth factor, growth factor receptor, hedgehog protein, hemoglobin, hepatocyte growth factor (HGF), hirudin, human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA- 1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon, IFN-α, IFN-ß, IFN-γ, interleukin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, Luciferase, neuroturin, neutrophil inhibitor (NIF), oncostatin M, bone morphogenetic protein, oncogene products, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, human growth hormone, playotropin, protein A, protein G, pyrogenic Toxin A, B or C, relaxin, renin, SCF, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigen, Staphylococcal Enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, Steroid Hormone Receptor, Superoxide Dismutase, Toxic Shock Syndrome Toxin, Thymosin Alpha 1, Tissue Plasminogen Activity Now, Tumor Growth Factor (TGF), TGF-α, TGF-ß, Tumor Necrosis Factor, Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Beta, Tumor Necrosis Factor Receptor (TNFR), VLA-4 Protein, VCAM-1 Protein, Vascular Endothelial growth factor (VEGF), urokinase, Mos, Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and / or corticosteroid.

Protein-protein conjugates including but not limited to heterodimers, heterotrimers, heteromultimers, homodimers, homotrimers, homomultimers, asymmetric homodimers, asymmetric homotrimers and asymmetric homodimers The crosslinking agent used in the formation of has a benzaldehyde, acetophenone and / or benzophenone residue at each end. Such residues react with the pyrrolysine and / or PCL residue (s) incorporated into the protein using the methods provided herein, and such proteins include those provided herein. A non-limiting example of a bifunctional crosslinking agent used to form homodimers is shown in FIG. 43A. This bifunctional linker was used to crosslink fibroblast growth factor 21 (FGF-21) (FGF21 Lys84PCL) with PCL residues incorporated at position 84. 43B is the mass spectrum of the reaction mixture, where the peak for the covalent FGF21 Lys48PCL dimer of expected mass 43037.2 Da is dominant. Unreacted FGF21 Lys84PCL is detected at 21235.2 Da, while some monomeric FGF21 modified at one end of the attached crosslinker is detected at 21820.0 Da. Reaction conditions were modified to fully react FGF21 Lys84PCL. In Figure 43 the protein conjugate linkage formed by this crosslinking is a quinazoline-type residue, but in other embodiments the linkage is a reduced form of the quinazoline-type residue (FIGS. 22, 23 and 30). In other embodiments, the linking is a fused ring residue (FIGS. 22, 23 and 30).

44A shows the mass spectrum of the reaction mixture, where the peak of the covalent FGF21 dimer of expected mass 43034 Da is dominant. Unreacted FGF21 Lys84PCL is not detected at 21234 Da, but some monomeric FGF21 modified at one end of the attached crosslinker is detected at 21818 Da. 43B is SDS-PAGE for reactions at pH 5.0 and pH 7.5, showing better conversion at pH 5.0. PCL-specific crosslinking was used to generate dimers of FGF21. However, the method is applicable to any of the proteins described above and can be used to form dimers, trimers and multimers. Such crosslinking is used to enhance the activity of the biologically active protein. In addition, this approach is used to stabilize complexes and / or stabilize receptor decoys for structural studies. 45 shows an embodiment using a crosslinker for trimer formation.

Site specific labeling

In another aspect using the methods provided herein, site specific incorporation of one or more pyrrolysine and / or PCL into proteins, polypeptides and / or peptides may be used to site specific such proteins, polypeptides and / or peptides. Labeling as an enemy. This labeling is due to the reaction of a label derivatized with a functional group that selectively reacts with pyrrolysine and / or PCL residue (s) and pyrrolysine and / or PCL residue (s). Labels used include dyes, antibodies or antibody fragments, metal chelating agents, spin labels, fluorophores, metal-containing residues, radioactive residues, actinic radiation-exciting residues, ligands, biotin, biotin analogues, redox-active agents, isotope labels Residues, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, insertable groups, chromophores; Energy transfer agents, quantum dot (s), fluorescent dyes, FRET reagents, and any combination thereof. The label is also any X 1 as defined herein.

In one embodiment, such site-specific labeling of such proteins, polypeptides and / or peptides containing one or more pyrrolysine and / or PCL residues may comprise benzaldehyde, acetophenone and pyrrolysine and / or PCL residue (s). Or a reaction derivatized with a benzophenone moiety. Labels used include dyes, antibodies or antibody fragments, metal chelating agents, spin labels, fluorophores, metal-containing residues, radioactive residues, actinic radiation-exciting residues, ligands, biotin, biotin analogues, redox-active agents, isotope labels Residues, biophysical probes, phosphorescent groups, chemiluminescent groups, electron dense groups, magnetic groups, insertable groups, chromophores; Energy transfer agents, quantum dot (s), fluorescent dyes, FRET reagents, and any combination thereof. The label is also any X 1 as defined herein.

In a further aspect of such site specific labeling provided herein, a label derivatized with a residue that selectively reacts pyrrolysine and / or PCL residue (s) with pyrrolysine and / or PCL residue (s). Reaction to increase detection sensitivity. Specifically, this reaction forms a detectable moiety that has different spectral properties than that of the reactant present before the reaction, thereby enhancing the ratio of signal to noise by minimizing background signals associated with the detectable moiety signal. In certain embodiments of such site specific labeling provided herein, the reaction of a pyrrolysine or PCL residue (s) with a label derivatized with benzaldehyde, acetophenone or benzophenone residues is performed by a benzaldehyde, acetophenone or benzophenone residue. Quinazoline-type residues with spectral properties different from those formed (FIGS. 22, 23 and 30). In certain embodiments of such site specific labeling provided herein, the reaction of a pyrrolysine or PCL residue (s) with a label derivatized with benzaldehyde, acetophenone or benzophenone residues is performed by a benzaldehyde, acetophenone or benzophenone residue. Reduced quinazoline-type residues having spectral properties different from those (Figs. 22, 23 and 30). In certain embodiments of such site specific labeling provided herein, the reaction of a pyrrolysine or PCL residue (s) with a label derivatized with benzaldehyde, acetophenone or benzophenone residues is performed by a benzaldehyde, acetophenone or benzophenone residue. To form fused ring residues having different spectral properties than those (FIGS. 22, 23 and 30).

In certain embodiments such labeled proteins, polypeptides and / or peptides are used for diagnosis and imaging, and in other embodiments such labeled proteins, polypeptides and / or peptides are used in high throughput screening. In other embodiments such labeled proteins, polypeptides and / or peptides are used to assess transport properties, and in other embodiments such labeled proteins, polypeptides and / or peptides are used in localization studies.

46 illustrates certain embodiments of such site specific labeling. 46A illustrates labeling with fluorescent moieties. 46B illustrates reacting PCL residues with non-fluorescent residues that fluoresce in reaction. 46C illustrates biotinylation of protein. 46D illustrates labeling with radioactive moieties. FIG. 46E illustrates labeling with 1- (2-amino-5-iodophenyl) ethanone, FIG. 46F illustrates labeling with 1- (2-amino-5-bromophenyl) ethanone, Both can be used to obtain phase information for the method of determining the X-ray crystal structure of a protein. 1- (2-amino-5-iodophenyl) ethanone may also be used for protein labeling with radioactive moieties. In Figure 46 the protein conjugate linkage formed by this labeling is a quinazoline-type residue, but in other embodiments the linkage is a reduced form of the quinazoline-type residue (FIGS. 22, 23 and 30). In other embodiments, the linking is a fused ring residue (FIGS. 22, 23 and 30).

In embodiments of such site specific labeling of such proteins, polypeptides and / or peptides containing one or more pyrrolysine and / or PCL residue (s), the precursor labeled pyrrolysine or PCL residue (s) itself Can be labeled. 46G and 46H show by way of example only labeling by radioactive or stable isotopes from differently labeled precursors.

Another aspect of the invention provides for the production of proteins homologous to any available protein but comprising one or more pyrrolysine or PCL residue (s). For example, in certain embodiments, a therapeutic protein is prepared that comprises one or more pyrrolysine or PCL and homologous to one or more therapeutic proteins. For example, in one aspect, the protein is homologous to, for example, the following therapeutic or other proteins: cytokines, growth factors, growth factor receptors, interferons, interleukins, inflammatory molecules, oncogene products, peptide hormones, signals Transduction molecules, steroid hormone receptors, transcriptional activators, transcriptional inhibitors, erythropoietin (EPO), fibroblast growth factor, fibroblast growth factor 21 (FGF21), leptin, insulin, human growth hormone, epidermal neutrophil activating peptide-78 , GROα / MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1, hepatocyte growth factor, insulin-like growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, playotropin, SCF, c-kit ligand, VEGF, G-CSF, IL-1, IL-2, IL-8, IGF-I, IGF-II, FGF (fibroblast growth factor), PDGF, TNF, TGF-α, TGF -ß, EGF (epidermal growth factor), KGF (keratinocyte growth factor), CD40L / CD40, VLA-4 / VCAM-1, ICAM-1 / LFA-1, hyaluronic / CD44, Mos, Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and / or corticosteroid. In another set of embodiments, the protein is homologous to, for example, the following therapeutic or other proteins: alpha-1 anti-trypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial sodium Diuretic factor, atrial natriuretic polypeptide, atrial peptide, CXC chemokine, T39765, NAP-2, ENA-78, Gro-α, Gro-β, Gro-γ, IP-10, GCP-2, NAP-4, SDF- 1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocytes Inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, C-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, GROα / MGSA, GROβ, GROγ, MIP-1α , MIP-16, MCP-1, epidermal growth factor (EGF), epidermal neutrophil activating peptide, interferon alpha (INF-α) or interferon beta (INF-β), exfoliating toxin, factor IX, factor VII, factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, G-CSF, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, hedgehog protein, hemoglobin, hepatocyte growth factor ( HGF), hirudin, human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon, IFN -α, IFN-ß, IFN-γ, interleukin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neuroturin, neutrophil inhibitory factor (NIF), on costintin M, bone morphogenetic protein, oncogene products, Parathyroid hormone, PD-ECSF, PDGF, peptide hormone, Liver growth hormone, fluorotrophin, protein A, protein G, pyrogenic exotoxin A, B or C, relaxin, renin, SCF, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen, Staphylococcus enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, Steroid hormone receptor, Superoxide dismutase, Toxicity Shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), TGF-α, TGF-ß, tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor ( TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, Mos, Ras, Raf, Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and / or corticosteroid.

In one aspect, the compositions herein comprise a protein comprising one or more pyrrolysine or PCL residue (s), such as any of the foregoing, and pharmaceutically acceptable excipients.

In certain embodiments the protein is a therapeutic protein, in other embodiments the therapeutic protein is erythropoietin (EPO), fibroblast growth factor 21 (FGF21), interferon alpha (INF-α) or interferon beta (INF-β) to be. In certain embodiments, the therapeutic protein is an antibody fragment, including but not limited to an antibody or a Fab. In certain embodiments, the therapeutic protein is produced as a fusion protein with modified fusion partners. In certain embodiments, the therapeutic protein is a fusion with an Fc domain.

Homology to a protein or polypeptide can be inferred by, for example, setting a default parameter to perform sequence alignment, eg, BLASTN or BLASTP. For example, in one embodiment, the protein is at least about 50%, at least about 75%, at least about 80 with known therapeutic proteins (eg, proteins present in Genebank or other available databases). %, At least about 90% or at least about 95% equal.

To demonstrate site specific labeling, PCL was incorporated into mEGF using the methods provided herein (see Example 12), and PCL residues were coupled to biotin through polyethers functionalized with ABA residues (X3626). -140, Example 40). 47A shows ESI mass spectrometric analysis of mEGF-Tyr10PCL conjugated with biotin (see Example 24). 47B shows western blotting of mEGF-Tyr10PCL-ABA-biotin conjugates using horseradish peroxidase (HRP) conjugated goat anti-biotin antibody. Uncoupled mEGF-Tyr10PCL and fluorescein-conjugated mEGF-Tyr10PCL served as negative controls.

To further demonstrate site specific labeling, PCL was incorporated into mEGF using the methods provided herein (see Example 12), and PCL residues were coupled to fluorescein via a polyether functionalized with ABA residues. Ring (see X3757-48, Example 41). 47C shows ESI mass spectrometric analysis of mEGF-Tyr10PCL conjugated with fluorescein (see Example 25). In addition, PCL was incorporated into mEGF using the methods provided herein (see Example 12) and PCL residues were coupled to disaccharides functionalized with ABA residues (see 3793-050, Example 42). 47D shows ESI mass spectrometric analysis of mEGF-Tyr10PCL conjugated to disaccharides (see Example 26). It is understood that the coupling chemistry used for the attachment of disaccharides can be readily applied to the coupling of other sugars and complex oligos and polysaccharides.

Coupling of immunomodulators to pyrrolysine and / or pyrrolysine analogous proteins incorporated into proteins, polypeptides and / or peptides and enhancing immunogenicity by such coupling

In another aspect provided herein, proteins, polypeptides and / or peptides having one or more pyrrolysine and / or PCL incorporated therein are derivatized with one or more immunomodulators. Using the methods provided herein, immune stimulating residues can be easily coupled to proteins, polypeptides and / or peptides via pyrrolysine and / or PCL residue (s). These immune stimulating moieties include one or more nitro groups, lipids, phospholipids, LPS-like molecules, adjuvants, adjuvants-like molecules, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T -Cell epitopes, keyhole limpet hemocyanin (KLH), immunogenic hapten, halogen, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups. In certain embodiments, immunomodulators attached to pyrrolysine and / or PCL are used to stimulate the immune response to self-antigens in a manner similar to p-nitro-phenylalanine incorporated into proteins (Gruenewald J, Tsao ML, Perera R, Dong L, Niessen F, Wen BG, Kubitz DM, Smider VV, Ruf W, Nasoff M, Lerner RA, Schultz PG, Immunochemical termination of self-tolerance, Proc Natl Acad Sci US A. 2008 Aug 12; 105 (32): 11276-80). In certain embodiments, self-antigens with immunomodulators attached to pyrrolysine and / or PCL are used as cancer vaccines. In other embodiments, the antigen with an immunomodulator attached to pyrrolysine and / or PCL is used as a vaccine for infectious disease. In other embodiments, the antigen with an immunomodulator attached to pyrrolysine and / or PCL is used as a vaccine against diseases involving the formation of amyloid, such as but not limited to Alzheimer's disease.

In one embodiment, a library of 2-aminobenzaldehyde compounds is generated, and after coupling the library to pyrrolysine or PCL incorporated into the antigen of interest, the coupled product is a high titer antibody against modified and unmodified antigens. Screening for the generation of yields a library of immune stimulating residues. In such embodiments, the 2-aminobenzaldehyde moiety comprises at least one nitro group, lipid, phospholipid, LPS-like molecule, adjuvant, adjuvant-like molecule, TLR2 agonist, TLR4 agonist, TLR7 agonist, TLR9 agonist With various substituents, including but not limited to, TLR8 agonists, T-cell epitopes, keyhole limpet hemocyanin (KLH), immunogenic haptenes, halogens, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups Is substituted.

In another embodiment, a library of 2-amino-acetophenone compounds is generated, and after coupling the library to pyrrolysine or PCL incorporated into the antigen of interest, the coupled product is then directed to the modified antigen and the unmodified antigen. Screening for the generation of high titer antibodies against produces a library of immune stimulating residues. In such embodiments, the 2-amino-acetophenone moiety comprises at least one nitro group, lipid, phospholipid, LPS-like molecule, adjuvant, adjuvant-like molecule, TLR2 agonist, TLR4 agonist, TLR7 agonist, TLR9 A variety of agonists, including but not limited to agonists, TLR8 agonists, T-cell epitopes, keyhole limpet hemocyanins (KLH), immunogenic haptenes, halogens, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups Substituted with a substituent.

In another embodiment, a library of 2-amino-5-nitro-benzophenone compounds is generated, and after coupling the library to pyrrolysine or PCL incorporated into the antigen of interest, the coupled product is modified into a modified antigen and Screening for the generation of high titer antibodies to unmodified antigens produces a library of immune stimulating residues. In such embodiments, the 2-amino-5-nitro-benzophenone moiety comprises one or more nitro groups, lipids, phospholipids, LPS-like molecules, adjuvants, adjuvants-like molecules, TLR2 agonists, TLR4 agonists, TLR7 Agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, keyhole limpet hemocyanin (KLH), immunogenic hapten, halogen, aryl groups, heteroaryl groups, cycloalkyl groups or heterocycloalkyl groups, including but not limited to Is substituted with a variety of substituents.

To demonstrate this aspect, PCL was incorporated into mTNF-α and mEGF using the methods provided herein (see Examples 11 and 12) and coupling the PCL residue to a hapten containing one or more nitrophenyl groups. (See Examples 27 and 28). 48A and 48B demonstrate the attachment of mono-nitrophenyl hapten (see 3793-001, Example 38-8) at the PCL incorporation site of mTNF-Gln21PCL (FIG. 48A) and mEGF-Tyr10PCL (FIG. 48B). 48C and 48D show the attachment of di-nitrophenyl hapten (TU3627-088, Example 38-7) at the PCL incorporation site of mTNF-α (FIG. 48C) and mEGF (FIG. 48D).

49A demonstrates the attachment of TLR7 agonists (see X3678-114, Example 38-3) at the PCL incorporation site of mEGF-Tyr10PCL (see Example 29). 49B demonstrates the attachment of phospholipids (see TU3627-092, Example 43-1) at the PCL incorporation site of mEGF-Tyr10PCL (see Example 31).

50-51 show PADRE peptides for mTNF-Gln21PCL or mEGF-Tyr10PCL: PX2-PADRE (MW = 1585) (see 3465-143; Examples 38-11) and BHA-exPADRE (MW = 2060) (3647-104 See Example 38-10) to demonstrate conjugation. 50A and 50B show MALDI-TOF mass spectrometric analysis of mTNF-Gln21PCL conjugation reaction with PX2-PADRE at two different pH values (see Example 30), and FIG. 50C shows mTNF-Gln21PCL conjugation with BHA-exPADRE ESI mass spectrometric analysis of the reaction is shown (see Example 30). 51 shows the coupling of BHA-exPADRE to mEGF-Tyr10PCL (see Example 30). The sequence of the PADRE peptide (SEQ ID NO: 28) is

Figure pat00065
Where X is cyclohexyl-alanine. The sequence of exPADRE peptide (SEQ ID NO: 29) is
Figure pat00066
Where X is cyclohexyl-alanine. In certain embodiments, known immunogenic peptide epitopes will be coupled with an antigen to enhance the immunogenicity of said antigen. In certain embodiments, the peptide from the antigen will be coupled with an immunogenic carrier protein. In certain embodiments, the immunogenic carrier protein is KLH. It is understood that the coupling chemistry used for the attachment of PADRE peptides can be readily applied to the coupling of other peptides to PCL and pyrrolysine.

Immuno- PCR : Coupling of DNA and Other Oligonucleotides to Pyrrolysine and / or Pyrrolysine Analogs Incorporated into Proteins, Polypeptides and / or Peptides

In another aspect provided herein, proteins, polypeptides and / or peptides having one or more pyrrolysine and / or PCL incorporated therein are derivatized into DNA using the methods provided herein. DNA is modified to have terminal 2-amino-benzaldehyde, 2-amino-acetophenone or 2-amino-5-nitro-benzophenone residues that react with pyrrolysine and / or PCL incorporated into proteins, polypeptides and / or peptides This DNA is coupled to a protein, polypeptide and / or peptide via a quinazoline-type linkage, a reduced quinazoline linkage or a fused ring linkage (see FIGS. 22, 23 and 30).

To demonstrate the attachment of DNA to PCL, PCL was incorporated into mTNF-α and mEGF using the methods provided herein (see Examples 11 and 12), and the PCL residue was also a CpG oligonucleotide that is also an immune stimulating residue. To (see Example 32). BHA-BG1 (see 3647-057, Examples 38-12) and BHA-BG2 (see 3597-167, Examples 38-14) were used to attach CpG to mTNF-Gln21PCL and mEGF-Tyr10PCL at the site of PCL incorporation. CpG reagent. Coupling of BHA-BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Gln21PCL (19.3 kDa) was confirmed by gel transfer assay (FIG. 52A) and BHA- to mEGF-Tyr10PCL (7.2 kDa). Coupling of BG2 (7.4 kDa) (FIG. 52B) was similarly confirmed. The sequence of BG1 (SEQ ID NO: 30) is

Figure pat00067
Where * represents a phosphothioate linkage. The sequence of BG2 (SEQ ID NO: 31) is
Figure pat00068
Where * represents a phosphothioate linkage. In certain embodiments, the oligonucleotide to be coupled will be deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid or other modified oligonucleotide. Initially coupled to pyrrolysine or PCL as a single stranded oligonucleotide, double stranded DNA, RNA, PNA or hybrid polymers can be readily prepared by hybridization with complementary strands. It is understood that the coupling chemistry used for attachment of CpG oligonucleotides can be readily applied to the coupling of other oligonucleotides.

In certain embodiments, the protein, polypeptide and / or peptide is an antibody, and immuno-PCR analysis is performed using such antibody to which DNA is linked. Immune-PCR is performed by linking DNA to an antibody using the methods described herein and then binding the DNA linked antibody to a target antigen. After binding, DNA is amplified by DNA amplification techniques and the resulting amplification products are analyzed by electrophoresis techniques, microplate methods or real-time PCR.

Site specific and orientation attachment

In another aspect provided herein, site-specific incorporation of one or more pyrrolysine and / or PCL to proteins, polypeptides and / or peptides is used to attach such proteins, polypeptides and / or upon attachment to a support surface. Control the direction of the peptide. This attachment is due to the reaction of pyrrolysine or PCL residues with surfaces derivatized with benzaldehyde residues, acetophenone residues, benzophenone residues, or a combination thereof, which results in the formation of quinazoline residue linkages and the orientation of the protein on the surface. . By incorporating pyrrolysine and / or PCL at specific locations in the protein, the direction of the protein at the surface is controlled. This control over the direction of protein attachment is used as a component of the protein engineering tool kit for assessing protein properties. FIG. 53 illustrates an embodiment of such site specific orientation attachment, wherein the surface is derivatized with 20 amino-acetophenone residues that react with PCL residues incorporated into the protein to allow said protein to be cleaved through a quinazoline-type linkage. Attach to the surface. In Figure 53 the protein conjugate linkage formed is a quinazoline-type residue, but in other embodiments the linkage is a reduced form of the quinazoline-type residue (see Figures 22, 23 and 30). In other embodiments, the linking is a fused ring moiety (see FIGS. 22, 23 and 30).

Non-limiting examples of supports include solid and semi-solid matrices such as aerogels and hydrogels, resins, beads, biochips (eg thin films coated with biochips), microfluidic chips, silicon chips, multi-well plates (also, And also referred to as microtiter plates or microplates), membranes, cells, conductive and non-conductive metals, glass (eg, microscope slides), and magnetic supports. Other non-limiting examples of solid supports used in the methods and compositions described herein include silica gel, polymer membranes, particles, derivatized plastic films, derivatized glass, derivatized silica, glass beads, cotton, plastic beads, Alumina gels, polysaccharides such as sepharose, poly (acrylate), polystyrene, poly (acrylamide), polyols, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, Mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride, polypropylene, polyethylene (e.g. poly (ethylene glycol)), nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, starch and the like do. In certain embodiments, the support used in the methods and compositions described herein is a support used for surface analysis, such as a surface acoustic wave device or a device using vanishing wave analysis, such as surface plasmon resonance analysis.

The surface of the solid support used for the attachment of proteins, polypeptides and / or peptides having pyrrolysine and / or pyrrolysine analogs incorporated therein may be hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cya Having reactive functional groups including, but not limited to, furnaces, amidos, ureas, carbonates, carbamates, isocyanates, sulfones, sulfonates, sulfonamides, and sulfoxides. Such functional groups include 2-amino-benzaldehyde residues, 2-amino-acetophenone residues and / or 2-amino-5-nitro-benzophenone residues that react with pyrrolysine or pyrrolysine analogs to form quinazoline residues. Used to covalently attach. In certain embodiments, such 2-amino-benzaldehyde residues, 2-amino-acetophenone residues, and 2-amino-5-nitro-benzophenone residues are selected from solid support reactive functional groups such as, but not limited to, hydroxyl, carboxylic, Coupled to a solid support by reaction with carboxylic, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide and sulfoxide It is part of the polymer linker.

In other embodiments, the surface of the solid support has streptavidin or avidin attached thereto and is used for attachment of proteins, polypeptides and / or peptides having pyrrolysine and / or pyrrolysine analogs incorporated therein, Wherein pyrrolysine and / or pyrrolysine analogs are used to site-specifically attach biotin to said proteins, polypeptides and / or peptides.

Other supports used in the methods and compositions described herein include resins used in peptide synthesis, for example, polystyrene, PAM-resin, POLYHIPE ™ resins, polyamide resins, poly (ethylene Glycol) -grafted polystyrene resins, polydimethyl-acrylamide resins and PEGA beads.

In certain embodiments, proteins, polypeptides and / or peptides having pyrrolysine and / or PCL incorporated therein are deposited in an array format on a solid support. In certain embodiments, such deposition is performed by ink jet technology utilizing piezoelectric and other forms of propulsion to move liquid from the small nozzle to the solid surface, or direct surface contact between the support surface and a delivery mechanism, such as a pin or capillary. . For contact printing, robotic control systems and multiplexed printheads allow for automated microarray fabrication. For non-contact deposition by piezoelectric propulsion techniques, the robotic system also allows for automated microarray fabrication using continuous or drop-on-demand devices.

Example

It is to be understood that the embodiments described herein and the following examples are for illustrative purposes only, and that various modifications or changes thereto will be apparent to those skilled in the art and are included within the spirit and scope of the present application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for the purposes indicated.

Example 1 Site Specific Incorporation of Biosynthesized PCL into Protein Using Mammalian Cells

This example provides a description of gene constructs that allow PCL incorporation into a TAG coding site in a target protein when transfected with mammalian cells. Although simultaneous incorporation at multiple sites is possible, all examples herein illustrate only PCL incorporation at a single site per protein molecule. This example also describes a general procedure for incorporating PCL into proteins using mammalian cells.

Construct:

Full length pylT, from the genomic DNA of Metanosarsina Majei Go1, using PCR using primers designed based on the pylS, pylT, pylB, pylC, and pylD nucleotide sequences of Metanosarsina Maze Go1 (NC_003901, see below) And the coding regions of pylS, pylT, pylB, pylC and pylD. The start codon of the genes was changed to ATG. pylT was cloned upstream of the CMV promoter into the pCMV vector. Transcription was controlled by the U6 promoter and formed the vector pCMVU6. pylS, pylB, pylC and pylD were similarly cloned into pCMVU6. Transcription of pylS, pylB, pylC and pylD was controlled by the CMV promoter (FIG. 4A).

The coding regions of the target genes human retinol binding protein (hRBP4), human and mouse erythropoietin (EPO) and mIgG1 Fc (mFc) were cloned into the pRS vector under CMV promoter control with the His tag at the C-terminus (FIG. 4B). Residues to be mutated with TAG codons were selected based on solvent-exposure in existing structural models as shown in Table 2. TAG codons were introduced into the target gene by PCR method.

Figure pat00069

4B shows the TAG mutant constructs of hRBP4, mEPO, hEPO and mIgG1. The mutation sites are listed in Table 2 and indicated by arrows in FIG. 4B. For each construct His tag was attached to the C-terminus as a purification and detection tool. Only full length proteins may have His tags. pylT, pylS, pylB, pylC and pylD were cloned into pCMVU6 under CMV control (FIG. 4A). These were used for cotransfection studies.

Cell Culture and Transfection:

HEK293F cells were grown under 5% CO 2 at 37 ° C. as a suspension in 293 Freestyle expression medium. One day before transfection, cells were divided into 0.7 × 10 6 cells / mL. Plasmid DNA was prepared with the Qiagen Maxi plasmid preparation kit. Transfection was performed by the PEI method. PEI is mixed in a ratio of 2: 1 with plasmid DNA in Opti-MEM, the DNA complex is added to HEK293F cells at 1 μg plasmid DNA per mL of cell culture and the cells are added for 5 days with 5 mM Cyc or D- Cultured in the presence of ornithine. When cells were transfected with various plasmids, the ratio of total amount of PEI: plasmid DNA was always 2: 1.

Protein Purification and Analysis:

After 4 days of transfection, the cell culture was centrifuged at 2000 g for 20 minutes and the medium was collected for purification of His tagged proteins. The medium was loaded onto a Ni-NTA column previously equilibrated with 20 mM Tris-HCl (7.5), 150 mM NaCl, containing 10 mM imidazole. The column was washed with the same buffer and eluted with elution buffer (20 mM Tris-HCl 7.5, 150 mM NaCl, 300 mM imidazole). Eluted proteins were assayed by the Bradford method and SDS-PAGE. In some cases, the media and purified proteins were analyzed by Western blotting using antibodies to His tags or proteins.

The sequence of the pyl gene cloned from the genomic DNA of Metanosarcine or Majey is shown below:

Figure pat00070

Figure pat00071

Example 2: The above portion to the mammal of an animal using the cells generated by biosynthetic PCL hRBP4 specific incorporation

This example demonstrates site specific incorporation of PCL at the TAG coding site in model protein human RBP4 (human retinol binding protein 4). hRBP4 is a secreted protein whose structure has been characterized and the solvent exposed residues in hRBP4 are defined.

hRBP4 was cloned into the pRS vector with the Flag-His tag at the C-terminus (FIG. 4A). Transient transfection was used to fully express hRBP4 in HEK293F cells. TAG codons were introduced at 9 positions in separate hRBP4 constructs as shown in Table 2 and the following sequences. Note that the individual TAG codons were introduced in the codons of the underlined amino acid residues.

Figure pat00072

Figure pat00073

Figure pat00074

Tyr108 and Phe54 are the retinol binding sites of hRBP4, but all other TAG mutations are at protein residues exposed to the solvent. When expressed in mammalian cells, termination of translation at these TAG sites produces a truncated protein without the C-terminal His tag. Thus, such truncated proteins will not be recognized by anti-His antibodies. Read-through translation will produce full length products with His tags. Thus, detection with anti-His antibodies functions as an indicator of transcription progression activity.

hRBP4 Expression of:

pRSRBP was co-transfected into HEK293F cells with pCMVpyS, pCMVpyB, pCMVpyC and pCMVpyD and the cells were grown in the presence of 5 mM D-ornithine as described in Example 1. Then, hist tagged hRBP4 was analyzed using Western blotting and anti-His antibody, and only full-length hRBP4 with PCL incorporated into the TAG codon was detected by Western blotting because truncated hRBP4 did not contain His tag. It became. Thus, the presence of full length hRBP4 demonstrates PCL incorporation. As shown in FIG. 6A, the anti-His antibody detected a band at 26 kDa, the expected size for full-length hRBP4, indicating that PCL was incorporated into the mutated TAG codon. PCL was incorporated at different rates in the nine hRBP4 constructs (Table 2), at higher rates in the # 2, # 6 and # 9 mutant constructs and at lower rates in the # 7 and # 8 mutant constructs. Protein yields for # 2, # 6 and # 9 were 4-8 mg / L and about 20% of the yield for wild type hRBP4. These proteins were purified and subjected to mass spectrometry (MS) analysis. The mass obtained for the protein was consistent with that expected for the incorporation of PCL, and parallel MS analysis confirmed PCL incorporation at the expected site (FIGS. 6A-11).

FIG. 6B shows the SDS-PAGE of hRBP4 produced in HEK293F cells in which PCL was incorporated into hRBP4, and FIG. 6C shows its mass spectrum. hRBP4 was purified from media of cotransfected HEK293F cells in the absence of D-ornithine (A, lane 1) or in the presence of D-ornithine (A, lane 2) and analyzed by SDS-PAGE. Arrows indicate full length hRBP4. Purified protein was analyzed by mass spectrometry (B): The mass observed was 23166.0 Da, similar to the expected mass 23168 Da.

hRBP4 Site-specifically incorporated into PCL Mass spectrometry detection:

500 mL cultures of HEK293F cells transfected with pylS, pylT, pylC and pylD genes, and DNA for hRBP4 mutant # 6 (Table 2: hRBP4 Phe122PCL) were grown in medium supplemented with 5 mM D-ornithine. The medium was run twice on a Ni-NTA column to capture all full length hRBP4 protein. The combined elution fractions contained 4.3 mg of total protein. The observed mass of this protein was 23166.8 Da (expected 23168 Da), which was consistent with the incorporation of a single PCL residue (data not shown). Sample aliquots were trypsin digested and subjected to LC-MS analysis. The MS / MS spectrum of Figure 7 is a peptide

Figure pat00075
(Wherein residue F * has a mass that matches the mass of PCL), which confirms site specific incorporation of PCL at the desired TAG site of residue 122. Peptide
Figure pat00076
The assignment for is described below:

m / z at 647.86

Figure pat00077
Assignment to (F * = PCL)

Monoisotopic Mass (calculated) of neutral peptide Mr: 1273.6819

Variable variant:

F7: PCL at F (F)

Ion Score: 60 Estimated: 0.0013

Match (bold): 22/74 fragment ions (using the 32 strongest peaks)

Figure pat00078

8 is a mass spectrometric analysis of trypsin digest of human RBP4 Phe122PCL (

Figure pat00079
TIC and EIC of 2+ ions of), showing the incorporation of PCL at the target Phe122TAG site. 9 shows mass spectrometric analysis of trypsin digests of human RBP4 Phe122PCL. The mass spectrum is at m / z 425.57 (3+) and 637.85 (2+) respectively.
Figure pat00080
3+ and 2+ precursors of (F * = PCL), which further illustrate the incorporation of PCL at the target Phe122TAG site.

Example 3: Detection of mammalian produced by the biosynthesis of cell lysates from animals PCL

This example demonstrates that PCL is biosynthesized in mammalian cells and detectable as free amino acids in lysates of such cells. Thus, this example suggests that PCL is incorporated like pyrrolysine and 20 other naturally occurring amino acids and that PCL incorporation is not the result of post-translational protein modification.

cell Melt  medium PCL Detection

60 mL cultures of HEK293F cells were grown with pylT, pylS, pylB, pylC and pylD genes in the presence or without 5 mM D-ornithine. The cells were harvested and lysed by sonication in 250 μl double-deionized water. Cell debris was pelleted by centrifugation. Cold methanol was added to a final concentration of 80% to precipitate the protein and remove by centrifugation. The soluble lysate was then concentrated by speed bag and analyzed by high pressure liquid chromatography in combination with mass spectrometry to confirm the presence of PCL.

The dried sample was first reconstituted in 100 μl of 98/2 mobile phase A / mobile phase B (mobile phase A: water with 0.1% formic acid; mobile phase B: acetonitrile with 0.1% formic acid). The sample was then diluted 20-fold and 2 μl injected into HPLC. Separation of analytes was performed on an Agilent Zorbax 300SB_C18 reversed phase HPLC column using the following solution gradient: 0 min 2% B; 5 minutes 2% B; 60 minutes 100% B; Flow rate: 0.25 mL / min. Comparison of the HPLC tracer (FIG. 10A) is present in lysates (bottom EIC (extracted ion chromatogram) tracer) of cells transfected with biosynthetic genes pylB, pylC and pylD and grown in the presence of D-ornithine The lysates of cells grown without D-ornithine (upper EIC tracer) show peaks at 4.13 min (indicated by asterisks) that are not present. The full scanning mass spectrum of the 4.13 min HPLC peak (FIG. 10C) shows a mass of 242.14943 Da, which is consistent with the theoretical mass (242.14992 Da) for PCL, a demethylated version of PYL. Lysine (HPLC peak at 1.44 minutes) is equally rich in both samples (FIG. 10B), and the total mass spectrum of lysine (FIG. 10D) serves as an internal correction and illustrates the accuracy of the method. This analysis shows that PCL is produced as free amino acid detectable from D-ornithine in cell lysate.

Example 4: Test using a mixed combination of different putative PCL and pyrrolidine new precursor, synthesis pyrrolidinyl new analogs, and biosynthesis gene

This example demonstrates that D-ornithine is the preferred precursor of PCL biosynthesis as measured by site specific incorporation into human RBP4. In addition, this example illustrates site specific incorporation of certain pyrrolysine analogs, such as CYC, at the TAG coding site in model protein human RBP4, human retinol binding protein 4, using mammalian cells. This example also provides insight into the substrate specificity of pylS tRNA synthetase.

N-ε- Cyclopentyloxycarbonyl -L-lysine ( CYC ) Mixing

DNA constructs for nine hRBP4 TAG mutants (Table 3) were individually transfected into HEK293F cells with pylT / pylS DNA as described in Example 2 and 4 mM N-ε-, a pyrrolysine analogue. Incubated without or in the presence of cyclopentyloxycarbonyl-L-lysine (CYC). Culture medium was harvested and analyzed by western blotting with anti-His and anti-hRBP4 antibodies (FIG. 11A). Western blotting with anti-His antibodies revealed 24 kDa protein for six of the nine hRBP4 TAG mutant constructs. The size of the protein corresponds to the size of wild type hRBP4, indicating the production of full length protein through transcriptional progression translation. Transcription progression activity was different per hRBP4 mutant constructs (Table 3) and was dependent on CYC and pylS. In particular, full length hRBP4 protein was not detected in mutants # 1, 5 and 7. High yield full length protein was observed in mutants # 2, 6 and 9, which was similar to the results seen for PCL incorporation (Example 2, FIG. 6A).

Figure pat00081

The expressed protein containing CYC was purified from the medium using Ni-NTA chromatography and analyzed by SDS-PAGE and then Coomassie blue staining. 11B shows SDS-PAGE analysis of purified hRBP4 TAG mutant # 2. The purified formulation showed a single protein band of 24 kDa size. Mass spectra of the purified protein were consistent with single site incorporation of CYC (FIG. 11C). The expected molecular weight of single site CYC incorporation in place of Phe62 was (23114.6 Da (wild type)-165.2 Da (Phe) + 258.3 Da (CYC)) 23208.7 Da, with an observation of 23182.0 Da. The observed mass suggests that the N-terminal Q residue is cyclized to pyrrolidone carboxylic acid resulting in 18 Da missing and 6 Da missing due to the presence of three intact disulfide bonds (23208.7 Da-6 Da-). 18 Da = 23184.7 Da). Parallel MS analysis of the purified protein demonstrates that CYC is incorporated at the designated TAG site in the hRBP4 construct (FIG. 12). Protein yield from transient transfection was estimated to be approximately 5 mg / L.

Figure pat00082
MS / MS fragmentation of is as follows:

Monoisotopic Mass (calculated) of neutral peptide Mr: 3248.5646

Variable variant:

F7: Cyc at F (F)

Q9: deamidation (NQ)

N11: Deamidation (NQ)

M24: oxidation (M) (neutral loss 0.0000) (shown in the table), 63.9983

Ion Score: 113 Estimated: 4.6e-009

Match (bold): 110/498 fragment ions (using 156 of the strongest peaks)

Figure pat00083

Presumptive PCL  And incorporation tests using pyrrolysine precursors

Production of full-length hRBP4 Phe122PCL protein (mutant # 6) was estimated by the putative PCL precursors D-ornithine, D-proline, D-arginine, D-glutamic acid, 4-hydroxyl-D-proline and 2-pyrrolidone- Determined in the presence of 5-carboxylic acid (FIGS. 13A and B, FIG. 14A). pRSRBP was co-transfected with HEK293F cells with pCMVpyS, pCMVpyB, pCMVpyC and pCMVpyD and the cells were grown in the presence of 5 mM of the putative precursor as described in Example 2 (FIG. 14A). Full length hRBP4 with His tag and PCL incorporation was then analyzed by Western blotting and anti-His antibody and SDS-PAGE. As shown in FIG. 13B, only D-ornithine produced a measurable protein band in SDS-PAGE of the Ni-NTA purified sample. Detection by Western blotting of the unpurified sample (FIG. 13A) showed that full-length hRBP4 protein was formed only at low levels in the presence of all other precursors, indicating that D-ornithine is the most efficient precursor for biosynthesis and incorporation of PCL Is clearly indicated. From Western blotting and SDS-PAGE analysis, the biosynthesis production of PCL from 5 mM D-ornithine (lane 2) and subsequent protein incorporation were known PylS substrates added at 5 mM to the medium of cells transfected with pCMVpyS. It is considered more efficient than the incorporation of CYC (lane 9). This example demonstrates that D-ornithine is the preferred precursor for the biosynthesis of PCL.

Incorporation tests using synthetic pyrrolysine analogs

Equivalent to the above experiments, production of full-length hRBP4 Phe122PCL protein (mutant # 6) was also determined in the presence of a series of synthetic pyrrolysine analogs designed to have acetyl residues for chemical derivatization after protein incorporation (FIG. 14B). . Synthetic analogs were prepared as described in Example 19. pRSRBP was co-transfected into HEK293F cells with pCMVpyS providing one gene copy of pylT tRNA and pylS tRNA synthetase. Depending on solubility, the synthetic analogues were added to the medium at a final concentration of 2 or 5 mM. Subsequently, detection of full-length hRBP4 with His tag and PCL incorporation was analyzed by Western blotting and anti-His antibody and SDS-PAGE. As shown in FIG. 13B, only CYC (lane 9) produced measurable protein bands in the analysis of Ni-NTA purified samples. Western blotting analysis of the unpurified sample (FIG. 13A) suggests that TU3000-016 (lane 15) is also an available substrate of pylS tRNA synthetase. However, the compound is unstable when the incorporation efficiency is low and the different batches (TU2982-150) are degraded and much less incorporated into hRBP4 when measured by nuclear magnetic resonance spectroscopy (lane 10). Consistent with previous published reports, this example demonstrates that pyrrolysine analogs characterized by sp2 carbons at ring residue attachment points are not acceptable as substrates of pylS tRNA synthetases.

Incorporation tests using various combinations of biosynthetic genes

Production of full-length hRBP4 Phe122PCL was also tested by co-transfection with cell cultures containing 5 mM D-ornithine and several combinations of biosynthetic genes pylB, pylC and pylD (FIG. 13C). All cultures were cotransfected with pCMVpyS providing one gene copy of pylT tRNA and pylS tRNA synthetase. Full-length hRBP4 protein is detected by Westin blotting with anti-His tag antibody only when both pylC and pylD are cotransfected. Although pylB is deemed necessary for PYL biosynthesis, it is not essential for PCL biosynthesis and subsequent incorporation. This observation is further confirmed by the production of the mIgG1 Fc domain protein (Example 5) and full-length PCL mutants of mouse and human EPO (Example 6). All three examples illustrate that only genes pylC and pylD are essential for PCL biosynthesis and protein incorporation.

Example 5 Site Specific Incorporation of Biosynthetically Generated PCL of Mouse IgG1 into the Fc Domain Using Mammalian Cells

This example demonstrates that site specific incorporation of biosynthetically generated PCL in mammalian cells using the methods provided herein is a general procedure and is not limited to protein hRBP4.

mIgG1 Fc Manifestation of

Four TAG mutants were generated in K333 (mutant # 1), K336 (mutant # 2), T394 (mutant # 3), and L426 (mutant # 4) of the Fc domain of mouse IgG1 (Table 2). And FIG. 17A). pRSFc # 1-4 (Table 2) was co-transfected into HEK293F cells with pCMVpyT, pCMVpyS, pCMVpyC and pCMVpyD and the cells were grown in the presence of 5 mM D-ornithine (pCMVpyB was not added). His tagged Fc domain protein was purified from the medium using Ni-NTA chromatography and analyzed by SDS-PAGE. The size of the protein bands in the gel for each construct matched their expected size for full length protein (FIG. 17A). Only the full length protein was recovered because the expression construct was characterized by a C-terminal His6 tag for purification. As FIG. 17A shows, expression of mutant # 1 depends on whether D-ornithine was added to the growth medium. The expression levels of all four mutants were similar. If the Fc domain is produced in HEK293F cells, no mass spectrometric analysis was performed because it is glycosylated.

Example 6 Site Specific Incorporation of Biosynthetic PCL into Erythropoietin (EPO) Using Mammalian Cells

In this example, PCL was site-specifically incorporated into erythropoietin, which further demonstrates that the method provided herein is a general procedure for site-specific incorporation of biosynthetically produced PCL into proteins in mammalian cells.

Mouse erythropoietin ( EPO ) Expression

PCL incorporation into the EPO mutant protein was performed in HEK293F cells. TAG mutations were introduced at 11 surface-exposed Lys or Arg residues away from the EPO receptor binding interface. The incorporation sites are shown below and listed in Table 2. Mouse EPO protein was expressed as described in Example 1 except that no pylB gene was used: pRSEPO # 1-11 (Table 2) was co-transfected into HEK293F cells with pCMVpyT, pCMVpyS, pCMVpyC and pCMVpyD. Infected and cells were grown in the presence of 5 mM D-ornithine. His tagged EPO was purified from the medium using Ni-NTA chromatography and analyzed by SDS-PAGE. The EPO construct contains a C-terminal His-tag. Therefore, only proteins in which PCL has been successfully incorporated will produce full length protein and will be purified by Ni-NTA chromatography, thus producing a detectable band in SDS-PAGE. SDS-PAGE of full-length mouse EPO indicates successful incorporation of PCL in all 11 mutants (FIG. 17B). The size of the protein bands in the gel for each construct matched their expected size for full length protein. For mutant # 1, it is illustrated that the formation of full-length protein depends on the presence of D-ornithine. The expression levels of all 11 mutant proteins were similar, ranging from 10% to 20% of wild type protein expressed at about 40 mg / L (FIG. 17B). Mass spectrometric analysis was not performed because EPO is glycosylated in HEK293F cells. The sequences of the mature human (SEQ ID NO: 19) and mouse (SEQ ID NO: 20) EPO proteins are shown below, the PCL incorporation sites are shown in bold and the glycosylation sites are underlined. Numbering of the mutants begins at the N-terminus (see also Table 2).

Figure pat00084

Example 7 Plasmids for Incorporating Biosynthetically Generated PCL into Proteins When Using Escherichia coli Cells

This example relates to a plasmid in which PCL can be incorporated into a TAG coding site in a target protein expressed from a second plasmid cotransformed with the same Escherichia coli cells when transformed into Escherichia coli cells. Provide a substrate.

pAra - pylSTBCD  build

this. A plasmid for incorporating PCL in E. coli cells is shown in FIG. 5 and constructed as follows: Cassette encoding pylT under the proK promoter was synthesized by amplification of the 3 ′ sequence of promoter, tRNA, and pSUP (using overlapping primers) It was. The three pieces were then mixed with terminal primers encoding restriction enzyme sites for ApaLI and XhoI to synthesize the entire insert. After digestion with ApaLI and XhoI, the cassette was ligated to the pSUP backbone prepared with the same enzyme to produce pSUP-pylT. M. Coding sequences for pylS, pylB, pylC and pylD from Majey were amplified from appropriately prepared pCMVU6 plasmids (Example 1) and inserted into pMH4 without tags (Lesley SA, Kuhn P, Godzik A, Deacon AM). , Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, and Stevens RC, "Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline", Proc Natl Acad Sci USA 2002; 99: 11664-11669). Each whole promoter-CDS-terminator was then amplified with a primer with various restriction enzyme sites.

Figure pat00085
pylS PCR product was digested with KpnI and SbfI and ligated with pSUP-pylT between KpnI and PstI sites to generate pAra-pylST. pylB, pylC and pylD products were digested with respective enzymes and ligated into plasmid backbones prepared with NdeI and KpnI. After confirming the plasmid products of these four ligations by sequencing and diagnostic PCR, the entire cassette was amplified with a primer to add KpnI sites to both ends of the pylD-pylB-pylC cassette. This was digested with KpnI and ligated to pAra-pylST at the KpnI site to produce the final pAra-pylSTBCD (FIG. 5). The final plasmid contains pylD, pylB, pylC and pylS, each of which is controlled by separate arabinose-induced and T7 hybrid promoters, each of which is downstream of the rrnB terminator (same as pMH4 content) and under the proK promoter has a pylT gene (similar to the pSUP / pSUPAR content with a single tRNA copy) (Cellitti et al., "In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy ", J Am Chem Soc. 2008 Jul 23; 130 (29): 9268-81].

Example 8 Site Specific Incorporation of Biosynthetically Generated PCL into FAS-TE Using Escherichia coli Cells .

This example provides a description of the incorporation of PCL into the TAG encoded site of human fatty acid synthetase (FAS-TE) expressed from a second plasmid co-transformed with the same Escherichia coli cells.

Thioesterase domain coding residues 2221-2502 of human fatty acid synthetase (FAS-TE) were expressed from pMH4 vectors with N-terminal tag (MGDSKIHHHHHHENLYFQG) (SEQ ID NO: 21) (Lesley SA, Kuhn P, Godzik A). , Deacon AM, Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, and Stevens RC, "Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline", Proc Natl Acad Sci USA 2002; 99: 11664-11669). TAG codons were mutated using PIPE cloning (Klock, HE, Koesema EJ, Knuth MW, and Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts", Proteins. 2008 May 1; 71 (2): 982-94)), which is described in Cellitti et al., "In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy ", J Am Chem Soc. 2008 Jul 23; 130 (29): 9268-81. Mutants tested for PCL incorporation were Leu2222TAG / Leu2223Ile and Y2454TAG (FIG. 18A). HK100 cells were co-transformed with pMH4-FAS-TE plasmid and pAra-pylSTBCD and selected on LB + Kan + Cm plates. Liquid cultures were induced after growing at 37 ° C. in TB (Sigma) + Kan + Cm supplemented with 5 mM D-ornithine (Sigma or Nova Biochem). When OD 595 = 0.8, cells were transferred to 30 ° C. and induced with 0.2% arabinose after 15-30 minutes. After induction, cells were grown for approximately 20 hours before harvesting by centrifugation. Cells were lysed by sonication in TBS + 5% glycerol (pH 8). Soluble protein fractions were purified according to manufacturer's protocol on Ni-NTA (Qiagen) chromatography. The yield was 46-80 mg / L for FAS-TE Leu2222PCL / Leu2223Ile and 155-186 mg / L for FAS-TE Tyr2454PCL, 50-80% of the yield of wild type protein. The molecular size of the protein as determined by molecular size and mass spectrometry in SDS-PAGE was consistent with the incorporation of PCL alone in the desired position (FIG. 18B).

The sequence of FAS-TE (SEQ ID NO: 22) with two PCL incorporation sites (bold and underlined) is shown below (for Leu2222PCL, residue Leu2223 is mutated to Ile2223):

Figure pat00086

Example 9 Site Specific Incorporation of Biosynthetically Generated PCL into FKBP12 Using Escherichia coli Cells .

This example describes the incorporation of PCL into the TAG encoded site of FKBP12 expressed from a second plasmid cotransformed with the same Escherichia coli cells.

FKBP12 was expressed from a pET vector (Novagen) with an N-terminal tag (MGSSHHHHHHLEVLFQGP) (SEQ ID NO: 23). TAG codons were introduced at the Ile90 site using PIPE cloning (Klock, HE, Koesema EJ, Knuth MW, Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts" , Proteins. 2008 May 1; 71 (2): 982-94). BL21 (DE3) cells (Invitrogen) were co-transformed with pET-FKBP12 and pAra-pylSTBCD and selected on LB + Kan + Cm plates. Liquid cultures were grown at 37 ° C. in TB + Kan + Cm supplemented with 5 mM D-ornithine. When OD 595 = 0.4, cells were transferred to 30 ° C. and induced with 1 mM IPTG 30 minutes later. The cells were grown for another 20 hours before harvesting. Cells were lysed and purified for FAS-TE. The purified protein was then dialyzed with TBS and cleaved with HRV-3C protease (2U per 1 mg FKBP12) for 48 hours at 4 ° C. to remove His tags. The cleaved material was flowed into a Ni-NTA column, collected, concentrated and passed through an S75 size exclusion column (GE Healthcare) for further purification. The final protein was further concentrated to 15-20 mg / ml and then crystallized. The yield of FKBP Ile90PCL was 120 mg / L (crude). Crystallized FKBP Ile90PCL was used to obtain the X-ray structure of PCL-containing protein. 19A-C show SDS-PAGE and mass spectrometric data and crystallization results for PCL biosynthesically incorporated into FKBP12. The mass obtained was 12085.6 Da, which is consistent with 12084 Da, the value expected for single site incorporation of PCL.

The sequence of FKBP12 (SEQ ID NO: 24) with PCL incorporation sites (bold and underlined) is shown below:

Figure pat00087

Example 10 Site Specific Incorporation of Biosynthetically Generated PCL into Fibroblast Growth Factor 21 (FGF21) Using Escherichia Coli Cells .

This example provides for the incorporation of PCL into 20 separate TAG encoded sites of human fibroblast growth factor, FGF21, expressed from a second plasmid cotransformed with the same Escherichia coli cells.

Fibroblast growth factor 21, FGF21, was expressed from a pSpeedET vector with an N-terminal tag (MGDSKIHHHHHHENLYFQG) (SEQ ID NO: 21) and coding residues 33-209 of translated human proteins (Klock, HE, Koesema EJ, Knuth MW, Lesley, SA, "Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts", Proteins. 2008 May 1; 71 (2): 982-94). TAG codons for PYL analog incorporation and subsequent PEG attachment were introduced at 20 individual positions of the FGF21 (SEQ ID NO: 25) aa33-209 constructs: Ser35, Gln39, Arg47, Gln56, Arg64, Asp74, Lys84, Lys87, Lys97 , Arg100, Arg105, His115, Arg124, Glu129, Lys150, Arg154, Leu167, Leu170, Leu181 and Gln184. The sequence of the FGF21 (33-209 aa) construct is shown below along with the site of incorporation (highlighted in bold and underlined) in 20 separate constructs.

Figure pat00088

HK100 or BL21 (DE3) cells were co-transformed with pSpeedet-FGF21 plasmid and pAra-pylSTBCD and selected on LB + Kan + Cm. Liquid cultures were grown as in Example 8 and 5 mM D-ornithine was added prior to induction. OD 595 for conversion to 30 ° C. was tested at 0.2-1.0 and subsequently induced with 0.2% arabinose or 1 mM IPTG at OD595 = 0.4-2.0 after 15-30 minutes. After induction cells were grown for approximately 20 hours and then harvested. Cells were lysed with 5% glycerol, 1% Triton X-114, or 2.5% deoxycholate in TBS. Insoluble pellets were then resuspended in TBS + 6M guanidine-HCl (pH 8). The protein was purified on Ni-NTA resin and refolded after eluting on or off the column. The tag was subsequently removed with TEV protease and the product was purified by Ni-NTA, ion exchange, and size exclusion chromatography.

Representative data showing incorporation of PCL into FGF21 is shown in FIG. 20. Preliminary expression yields are reported in Table 4. Both full length and truncated FGF21 proteins were purified as the constructs were characterized as comprising N-terminal His-tags. The range in which the TAG codon was used as a stop codon (generating truncated protein) and the range used to incorporate PCL (generating full length protein) was dependent on different incorporation sites (FIG. 20). The expected yield of the full length protein of interest is 4 to 56 mg / L, but no FGF21 protein could be detected in three of the mutants (Table 4), and the total yield of the protein obtained in the remaining 17 mutants was 5.7. To 143 mg / L. PCL incorporation was confirmed by mass spectrometry for all mutants.

Figure pat00089

Example 11: mTNF by PCL Incorporation .

To express the mTNF-α Gln21PCL mutant, E. E. coli BL21 (DE3) cells were co-transformed with individual mutant mTNF-α genes on the pAra-pylSTBCD and pET22b plasmid vectors. Transformed cells were grown at 37 ° C. in TB medium in the presence of 5 mM D-ornithine and induced with 1 mM IPTG and 0.2% (w / v) arabinose when the OD 600 reached 0.5. The cells were then shaken and harvested at 30 ° C. for 12-16 hours. Cell pellets were stored at −20 ° C. until use. The X-ray crystal structure of mTNF-α was determined by the PCL incorporation sites Lys11 and Gln21 as shown below in the protein sequence of recombinant mTNF-α containing the N-terminal His 6 tag followed by the factor Xa cleavage site (GIEGR). Indicated:

Figure pat00090

Example 12: mEGF incorporated by PCL.

To express the mEGF-Tyr10PCL mutant, E. E. coli BL21 (DE3) cells were co-transformed with individual mutant mEGF genes on the pAra-pylSTBCD and pET22b plasmid vectors. Transformed cells were grown at 37 ° C. in TB medium in the presence of 5 mM D-ornithine and induced with 1 mM IPTG and 0.2% (w / v) arabinose when the OD 600 reached 0.5. The cells were then shaken and harvested at 30 ° C. for 12-16 hours. Cell pellets were stored at −20 ° C. until use. The X-ray crystal structure of mEGF showed the PCL incorporation sites Tyr10 and Tyr29 as shown below in the protein sequence of recombinant mEGF with the C-terminal His 6 tag (SEQ ID NO: 27):

Figure pat00091

Example 13: mTNF to the new pyrrolidine (Pyl), or incorporation of the PCL.

To insert PYL into mTNF-α, E. coli. E. coli BL21 (DE3) cells were transfected with the codon for Gln21 (CAA) to the stop codon (TAG) as well as the mutant mTNF-α gene on the pET22b plasmid vector, as well as M. mazei pylS, pylT, pylB, Co-transformation with pAra-pylSTBCD containing pylC, and pylD. To incorporate PCL alone into the mutant mTNF-α, pAra-pylSTBCD was replaced with pAra-pylSTCD lacking the gene for putative methyl transferase PylB. Transformed cells were grown at 37 ° C. in TB medium in the presence of 5 mM D-ornithine and induced with 1 mM IPTG and 0.2% (w / v) arabinose when the OD 600 reached 0.5. The cells were then shaken and harvested at 30 ° C. for 12-16 hours. Cell pellets were stored at -20 ° C. After thawing the cell pellet on ice for 15 minutes, the cells were resuspended in lysis buffer (20 mM Tris / HCl, 50 mM NaCl, pH 8.0) at 3 ml per gram wet weight. Lysozyme was added at 1 mg / ml and the cells were sonicated for 2 minutes on ice. Lysates were centrifuged at 30,000 × g for 20 minutes at 4 ° C. to pellet cell debris. 1 ml of 50% Ni-NTA slurry (Qiagen) was added to the cleared lysate and mixed gently by shaking at 4 ° C. for 60 minutes. The lysate-Ni-NTA mixture was loaded into the column and the passthrough was collected. The resin was washed with 20 mL of 25 mM imidazole in PBS (pH 8.0), then the protein was eluted with 2.5 ml of 250 mM imidazole in PBS (pH 8.0) and by using a PD-10 column (GE Healthcare). Buffer exchange with PBS, pH 7.4. Expression of mTNF-α Gln21TAG with or without pylB, as well as with or without D-ornithine was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 21A). In Figure 21A, lane 1 is the molecular weight of the standard SeeBlue plus 2 prestained standard; Lane 2 is expression in the presence of both pylB and D-ornithine; Lane 3 expression in the presence of pylB without D-ornithine; Lane 4 expression in the absence of pylB with 5 mM D-ornithine added; Lane 5 is expression in the absence of both pylB and D-ornithine. The data show that expression of full-length mTNF-α is dependent on the presence of D-ornithine and is similar in the absence or presence of the pylB gene.

Example 14 Incorporation of Pyrrolysine ( Pyl ) or PCL into mEGF

this. E. coli BL21 (DE3) cells were co-transformed with the individual mutant mEGF Tyr10TAG and Tyr29TAG genes on the pAra-pylSTBCD and pET22b plasmid vectors. Both constructs were expressed at 37 ° C. in TB medium in the presence of 5 mM D-ornithine and 1 mM IPTG and 0.2% (w / v) arabinose were added when the OD 600 reached 0.5. The temperature was then lowered to 30 ° C. and cells were harvested 16 hours after induction. The cell pellet was resuspended in 20 mL of 20 mM Tris / HCl, pH 8.5 and sonicated for 5 minutes. After centrifugation at 30,000 × g for 20 minutes, the supernatant was decanted. The pellet was resuspended in 20 mL of 20 mM Tris / HCl (pH 8.5) containing 2% (v / v) Triton-X100 by sonication. After another round of centrifugation at 30,000 × g for 20 minutes, the pellet was sonicated in 10 mL of 8 M urea, 20 mM Tris / HCl, 10 mM β-mercaptoethanol, pH 8.5. Insoluble cell debris was removed by centrifugation (30,000 xg, 20 min) and the supernatant was refolded in buffer (100 mM Tris / HCl, 4 mM reduced glutathione, 0.4 mM glutathione oxide, 20% (v / v) ethanol, pH). 8.5). The diluted sample was then dialyzed overnight at 4 ° C. against the refold buffer using a slide-a-riser dialysis cassette (3500 Da Molecular Weight Cutoff, Pierce). Insoluble protein was removed by centrifugation at 30,000 × g for 20 minutes and the supernatant was supplemented with β-mercaptoethanol to a final concentration of 2 mM. 1 ml of 50% Ni-NTA slurry (Qiagen) was added to the refolded protein and shaken gently at 4 ° C. for 60 minutes to mix gently. The protein-Ni-NTA mixture was loaded on the column and the passthrough was collected. The resin was washed with 20 mL of 25 mM imidazole in PBS (pH 8.0), then the protein was eluted with 2.5 ml of 250 mM imidazole in PBS (pH 8.0). Finally, the protein was buffer exchanged into PBS (pH 7.4) by using a PD-10 column (GE Healthcare). The purity of the protein samples was examined by SDS-PAGE (FIG. 21B). In Figure 21B, lane 1 is the molecular weight of the standard SeaBlue Plus2 prestained standard and lane 2 is the mEGF Tyr10TAG mutant protein after Ni-NTA purification.

In addition, mEGF Tyr10TAG, ESI-MS spectra were obtained for proteins obtained with or without PylB expression, as obtained using the method for mTNFα (FIG. 21C). The bottom mass spectrum in FIG. 21C shows that PYL incorporation occurs predominantly in the presence of the pylB gene (estimated mass of mEGF Tyr10Pyl = 7310 Da), and the top mass spectrum in FIG. 21C shows that the only PCL incorporation occurs in the absence of pylB. (expected mass of mEGF Tyr10Pyl = 7296 Da). As such, of the proteins observed in FIG. 21B, lane 2 was mEGF incorporating PYL (identified by additional LC-MS / MS analysis). Similarly, of the proteins observed in FIG. 21A, lane 2 is probably mTNFα with PYL incorporated and lane 4 is probably mTNFα with PCL incorporated.

In addition, PCL / PYL from LC-MS / MS extracted ion chromatogram mass spectrometry of PCL and PYL incorporation into mEGF Tyr10TAG samples expressing all genes (M. maze pylS, pylT, pylB, pylC, and pylD). Quantification of the proportions showed that PYL was 5 to 10 times richer than PCL, whereas LC- of PCL and PYL incorporation into mTNF Gln21TAG samples expressing all genes (M. maze pylS, pylT, pylB, pylC, and pylD). Quantification of the PCL / PYL ratio from MS / MS extracted ion chromatogram mass spectrometry showed that PCL was about 7 times richer than Pyl. Quantification in the absence of the PylB gene showed only PCL protein, indicating that PYL incorporation is absolutely dependent on pylB, further suggesting that PylB is indeed the methyltransferase required for Pyl biosynthesis.

Example 15 Incorporation of Other Analogues and Precursors

HK100 cells (derived from Genehogs; Invitrogen) were co-transformed with pAra-pylSTBCD, pAra-pylSTCD, pAra-pylSTC, pAra-pylSTD, or pSUPAR-pylST and pMH4-FASTE-L2222TAG-L2223I. . The cells were grown in a 25 ml culture of Terrific Broth (TB) (Sigma) at 37 ° C. to OD 595 about 0.6. Cells were transferred to 30 ° C. and analog or precursor compounds were added to each individual culture at the concentrations shown in FIG. 15. Compounds evaluated were N-ε-cyclopentyloxycarbonyl-L-lysine (CYC; Sigma), D-ornithine (Chem-Impex), PCL-A (see Example 36-1, Compound 3647-125) , PCL-B (see Example 36-2, see compound 3793-011), P2C (see Example 36-2, see compound 3647-164), P5C (see Example 35-1, see compound 3793-007) and Lys- Nε-D-ornithine (see Example 35-2, compound 3793-031). The cells were then induced with 0.2% arabinose 20 minutes later. After 18-20 hours, cells were harvested by centrifugation. Cells are lysed by sonication and purified on Ni-NTA (Qiagen) under natural conditions and evaluated by SDS-PAGE gel followed by Coomassie staining (0.25 ml Ni-NTA, 0.75 ml elution, 20 μl on gel). It was. 15A shows purified protein (FAS-TE) from cells grown in the presence of the indicated pyl gene (pylS / pylT or pylB / pylC / pylD / pylS / pylT) and fed the indicated compounds. Cyc and D-ornithine (D-Orn) were used as positive controls. No compound added was shown as negative control. Gel sample doses in lanes 1-11 and 12-18 were internally consistent, respectively.

Lanes 2 and 3 only show proteins obtained from cells grown with the pylS and pylT genes and PCL-B (Lys-P2C) or PCL-A (Lys-P5C), respectively. Both compounds were incorporated into the protein, showing that PylS can use both compounds. Lanes 5-11 show precursor Lys-Nε-D-Orn (lane 10) in cells containing the full set of pyl biosynthetic genes (pylB / pylC / pylD / pylS / pylT), or precursors with only pyrroline rings: P2C ( Lanes 6) and protein biosynthesis in the presence of P5C (lane 8) are shown. Precursors with only pyrroline rings: P2C (lane 6) and P5C (lane 8) were not sufficient to support PCL biosynthesis, suggesting that they are not intermediates of the pathway. However, Lys-Nε-D-Orn (lane 10) expressed a protein incorporating PCL in the amber codon, indicating that it is an intermediate of the PCL biosynthetic pathway. Table 5 shows the amount of protein obtained (Bradford), the observed mass and the ratio of PCL: PYL: CYC obtained.

Figure pat00092

To determine if Lys-Nε-D-Orn is an intermediate upstream of the biosynthetic pathway of any of the genes (pylC and pylD) required, HK100 cells (derived from Genegenes; Invitrogen) were selected from pSUPAR-pylST, pMH4 Co-transformed with -FASTE-L2222TAG-L2223I, and pAra-pylSTB, pAra-pylSTC, pAra-pylSTD, pAra-pylSTCD or pAra-pylSTBCD. 15B showed that only pylD (lane 15) was required to form PCL from Lys-Ne-D-Orn, suggesting that this is an upstream intermediate of pylC in the biosynthetic pathway. In addition, NMR evaluation confirmed that Lys-Ne-D-Orn is a substrate of PylD.

Example 16 Derivatization of 2-amino benzaldehyde , 2-amino- acetophenone and 2-amino-5-nitro-benzophenone of PCL incorporated in hRBP4 .

This example provides for labeling PCL with 2-amino-benzaldehyde (2-ABA), 2-amino-acetophenone and 2-amino-5-nitro-benzophenone. The reaction appears to follow the general scheme shown in FIG. 22. The formed structures were evaluated using mass spectrometry and NMR. FIG. 23 shows protein conjugates formed from three different 2-amino-benzaldehyde residues, providing expected mass changes and observed mass changes.

hRBP4 2- ABA Site-specifically modified by PCL Mass spectrometry detection

Retinol binding protein (hRBP4) was expressed in HEK293F cells as described in Example 2. PCL was incorporated instead of Phe122 (hRBP4 mutant # 6), which was confirmed by mass spectrometry (FIGS. 6A-11). 10 μl of hRBP4 Phe122PCL stock solution was mixed with 89 μl of 200 mM sodium acetate buffer (pH 5.0) and 1 μl of 1 M 2-ABA solution and incubated for 16 hours at room temperature. Final concentration in the reaction mixture was 17 μM hRBP4 Phe122PCL protein and 10 mM 2-amino-benzaldehyde (2-ABA). The mass spectrum of derivatized hRBP4 is shown in FIG. 24, with the mass obtained corresponding to the 2-ABA adduct of PCL (23269.2 Da). Unmodified hRBP4 had a mass of 23166.8 Da, so the observed mass increase of 102.4 Da (estimated + 103 Da) indicates that hRBP4 was modified by 2-ABA. The peak intensity of at least 96% in the mass spectrum is due to the 2-ABA adduct of PCL, indicating that the reaction is almost complete.

LC-MS analysis of trypsin digestion of 2-ABA-derivatized hRBP4 Phe122PCL protein was performed, confirming the YWGVASF * LQK peptide (SEQ ID NO: 17) expected by MS / MS analysis (FIG. 25A), where F * is shown in FIG. Having a mass that matches the mass of the 2-ABA-modified PCL as shown in Figure 23). The reaction was complete as no derivatized PCL residues were detected. The assigned MS / MS spectrum of YWGVASF * LQK (F * = PCL-2-ABA adduct) is given below:

Figure pat00093

25B is TIC and EIC of 2+ ions of YWGVASF * LQK (F * = PCL and PCL-2-ABA adducts), derivatized and derivatized EIC (extracted ion chromatogram) (not detected) ) Compared to the species to indicate that the reaction is complete. 25C is a mass spectrometric analysis of hRBP4 Phe122PCL derivatized with 2-ABA, where 3+ and 2+ precursors of YWGVASF * LQK were found at m / z 459.92 (3+) and 689.37 (2+), respectively. (F * = PCL-2-ABA adduct). This example demonstrated that the reaction with 2-ABA observed was site specific and incorporated into the desired TAG site of residue 122.

pH Reaction efficiency as a function of:

Reaction efficiency as a function of pH, (FIG. 26) shows that the 17 μM hRBP4 PCL mutant protein is reacted with or without reacting with 10 mM 2-amino-benzaldehyde (2-ABA) in 200 mM sodium acetate buffer (pH 5.0), Or by reaction with 10 mM 2-amino-benzaldehyde (2-ABA) 10 × PBS buffer (pH 7.4) at room temperature for 12 hours. Mass spectra of the reaction mixture showed that a total peak intensity of at least 87% corresponds to the peak intensity (+102 Da as expected) of the protein modified by one 2-ABA residue (FIG. 26A, B and C). . The pH 7.4 reaction mixture contained approximately 13% of unreacted protein (FIG. 26C), whereas only 4.2% of unreacted protein was detected in the reaction at pH 5.0 (FIG. 26B), which was at pH 5.0 compared to pH 7.4. It suggests that PCL is slightly more reactive. Since hRBP4 incorporating OMePhe instead of Phe62 was not modified by the presence of 10 mM 2-ABA, the reaction is specific to the presence of PCL (FIGS. 26D, E and F).

Reaction efficiency as a function of protein concentration of the reactants and reaction with other 2- ABA -like residues :

17 μM hRBP4 PCL mutant protein was added to 0.1 mM 2-amino-benzaldehyde (2-ABA), 0.1 mM 2-amino-acetophenone (2-AAP) or 0.1 mM 2-amino in 200 mM sodium acetate buffer (pH 5.0). Reaction with -5-nitro-benzophenone (2-ANBP) to assess the reaction efficiency as a function of the protein concentration ratio of the reactants and the reactivity with 2-ABA-like reactants. After 12 hours at room temperature, the mass spectrum of the reaction mixture showed the expected mass of the conjugated protein (FIG. 27). For 2-ABA, the relative intensity of correctly conjugated peaks was 88% of the total intensity; Only 4.2% of the protein remained unreacted (FIG. 27A). For 2-AAP, 93% appeared to have reacted; 4.5% did not react (FIG. 27B). For 2-ANBP, only 5.4% reacted (FIG. 27C), which seems to be due to low solubility of the reactive reagent, ie 2-ANBP precipitates immediately upon addition to the solution containing hRBP4 PCL mutant protein.

This example shows that the PCL modified protein reacts with other 2-amino-benzaldehyde analogs and is derivatized at the site where PCL is incorporated. In all cases, the mass measured for the modified protein was consistent with the mass expected in the structure shown in FIG. 23. The data also showed that the conjugation reaction proceeds with high efficiency and is nearly complete at only a low 6 to 1 reactant to protein ratio. The data also showed that each protein sample was derivatized only once. However, very high reactant to protein ratio (4700 fold) and multiple reactant molecules were observed to attach at pH 7.5 (FIG. 28A). Precipitation of the same sample at pH 5.0 also indicates that there are multiple reactions. Similarly hRBP4 (6.5 μM) modified with OMePhe instead of Phe62 showed a conjugation pattern similar to that observed in FIG. 28A when reacted with a 15400-fold molar excess of 2-ABA at pH 7.5 (FIG. 28B). This indicates that attachment under these conditions (more molar excess of reactant than protein) is not dependent on the presence of the PCL side chain, but will probably involve lysine side chains. However, the example reaction shows that the PCL residues incorporated into the protein can be specific and almost quantitatively derivatized by reaction with a 2-amino-benzaldehyde containing molecule added in a small molar excess.

Example 17 : Derivatization of 2-amino-acetophenone of PCL in FAS - TE

16 μM of FAS-TE Tyr2454PCL produced in Escherichia coli was reacted with 1 mM 2-AAP at room temperature for 16 hours as in Example 8 and at pH 5.0 at 4 ° C. for 24 hours in 200 mM sodium acetate buffer. I was. FIG. 29A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL and FIG. 29B shows the mass spectrum of the reaction mixture: 100% of the observable peak intensity is 116.8 Da larger than the mass of unreacted material, 2-AAP modified It occurred at 33318.8 Da, which is within the error of 116 Da mass increase expected in FAS-TE Tyr2454PCL. Similarly, at pH 7.4 the reaction was 95% complete (FIG. 29C (unreacted) and FIG. 29D (reaction)).

Example 18: in the PCL incorporated in hRBP4 2- amino-acetophenone-derivative by PEG8 Chemistry

This example shows that PCL incorporated into hRBP4 can be fully derivatized at a single site by polyethylene glycol (PEG) derivatives of 2-amino-acetophenone. In this example, PEG contains 8 ethylene glycol units and its structure is shown in FIG. 31. This example also shows that at pH 5.0 and pH 7.5, wild type hRBP4 does not react with 2-AAP-PEG8 with up to 2300 reagents for protein ratio.

hRBP4 mutant # 6 was prepared as in Example 2. 2-Amino-acetophenone PEG8 (TU3205-044) was prepared as described in Example 20. After allowing the reaction to proceed for 14 hours at room temperature and 72 hours at 4 ° C., mass spectra of the reaction mixture were obtained.

When the reaction was carried out at pH 7.5, 10 μl of hRBP4 Phe122PCL (0.22 mg / mL, pH 7.5 in PBS) was diluted with 10 μl of 10 × PBS. 0.2 μl or 2 μl of 100 mM stock solution of 2-AAP-PEG8 (in water) was added at final concentrations of 1 and 9.1 mM, respectively. Protein concentrations were 4.7 μM and 4.3 μM, respectively, and the reactions resulted in 210 or 2100 molar excess of protein. 32A shows the mass spectrum of the reaction mixture at 210 to 1 ratio, indicating that the reaction was approximately 95% complete, and the observed mass increase of 556 Da was identical to that expected (FIG. 31). 100% completion was obtained in the reaction at 2100 molar excess (data not shown).

Similarly, the reaction was performed at pH 5.0. In this case, 10 μl of hRBP4 Phe122PCL (0.22 mg / mL, pH 7.5 in PBS) was diluted with 90 μl of 200 mM sodium acetate buffer (pH 5.0). 1 μl or 10 μl of 100 mM stock solution of 2-AAP-PEG8 (in water) was added at final concentrations of 1 and 9.1 mM, respectively. Protein concentrations were 0.94 and 0.86 μM, respectively, and the reactions resulted in 1050 or 10500 molar excess of protein. 32B shows the mass spectrum of the reaction mixture at 1050 to 1 ratio. 100% completion was obtained in both reactions, and the mass increase of 556 Da observed was the same as expected (FIG. 31).

To test the reactivity of 2-AAP-PEG8 with wild type hRBP4 protein, the test response was set similar to the above. For pH 7.5 reactions, the final wild type protein concentration was 20 μM and the 2-AAP-PEG8 concentration was 1 or 9.1 mM, resulting in a molar ratio of 46 and 460 to 1. pH 5 reactions were performed at 4 μM protein concentration and 1 and 9.1 mM 2-AAP-PEG (230 and 2300 to 1 reactant to protein). In all four reactions, only unmodified wild type hRBP4 protein was observed at the expected mass (FIGS. 32D-F). This example shows that the coupling reaction of 2-AAP-PEG is very specific for the presence of PCL residues in the target protein.

Example 19 Derivatization of 2-Amino-acetophenone- PEG of various molecular weights of PCL incorporated into FAS - TE

This example shows the universality of the reactivity of the PCL side chain with 2-amino-acetophenone PEG. This example further shows that sufficient length of 2-AAP-PEG useful for modifying the biotherapeutic protein can be conjugated to the PCL modified protein.

16 μM of FAS-TE Tyr2454PCL produced in Escherichia coli as described in Example 8 was added at pH 5.0 at room temperature for 16 hours in 1 mM TU3205-044 (2-AAP-PEG8) and 200 mM sodium acetate buffer at room temperature. Reacted. 33A shows the mass spectrum of unreacted FAS-TE Tyr2454PCL and FIG. 33B shows the mass spectrum of the reaction mixture: In FIG. 33B, 100% of the observable peak intensity is at 33758.4 Da, which is 556 Da greater than the mass of the unreacted material. Occurred. This mass difference is as expected for the 2-AAP-PEG8 modified FAS-TE Tyr2454PCL (FIG. 31).

In a further example, PCL incorporated into Tyr2454 of FAS-TE was derivatized with three different 2-AAP-PEGs with MW of 0.5 kDa, 2.4 kDa and 23 kDa. FAS-TE single site PCL mutants were generated in Escherichia coli in a yield of approximately 160 mg / L (corresponding to a yield of about 80% by weight) as described in Example 8. Aliquots of FAS-TE Tyr2454PCL (0.16 mM in PBS, pH 7.5) were added to TU3205-044 (0.5 kDa 2-AAP-PEG), TU3205-048 (2.4 kDa 2-AAP-PEG) and TU3205-052 (23 kDa 2 -AAP-PEG) at a molar ratio of 10: 1 or 100: 1 for 6 days at 4 ° C or room temperature. The structure of PEG and its synthesis is described in Example 37.

Prior to SDS-PAGE analysis (FIG. 34), His-tagged FAS-TE protein was bound to Ni-NTA beads to remove excess PEG reagent, and the beads were washed repeatedly with PBS buffer. For gel and mass spectrometric analysis, excess buffer was removed from Ni-NTA beads and the protein was eluted with imidazole buffer. Specifically, 50 μl of Ni-NTA beads were added to 50 μl of reaction mixture, incubated for 2 hours, and the reaction mixture and buffer were separated by centrifugation. The beads are then washed three times with 1 mL PBS; 70 μl of 250 mM imidazole buffer, 20 mM Tris, pH 8 was added to the beads to elute the protein for gel and mass spectrometric analysis. PEGylation with 0.5 kDa 2-AAP-PEG could not be analyzed by SDS-PAGE, but confirmed by mass spectrometry. The extent of the reaction was approximately 57% for the 100: 1 room temperature reaction and 43% for the 100: 1 4 ° C reaction (data not shown). For larger 2-AAP-PEG, PEGylated products could be analyzed by SDS-PAGE (FIG. 34). All reactions were incomplete and progressed approximately 25-30% for 2.4 kDa and 23 kDa 2-AAP-PEG. The extent of single site conjugation and reaction at the PCL site was confirmed by mass spectrometry for 2.4 kDa 2-AAP-PEG (FIG. 33). Mass spectrometric data on 23 kDa 2-AAP-PEG derivatized proteins could not be obtained because the PEG was not homogeneous.

The reported reaction yield is a lower estimate because the stringency of Ni-NTA bead extraction can aid extraction of unreacted FAS-TE and PEGylation can lower the affinity of His-tags (PEG of FAS-TE Leu2222PCL). Is the case of data; data not shown). However, detection of PEGylated material after extraction showed that PCL-2-AAP-PEG ligation was stable.

Example 20 Derivatization of 2-amino-acetophenone- PEG of different molecular weights of PCL incorporated into FGF21

This example shows that FGF21 incorporating PCL at various positions can be PEGylated with 2-AAP-PEG reagent. This example also shows that PEGylated FGF21 mutants can be isolated from unreacted full-length FGF21 and truncated FGF21 by a combination of ion exchange and size exclusion chromatography.

FGF21 incorporating PCL at position lysine 84 was expressed in Escherichia coli, refolded and purified as described in Example 10 except that the protein did not cleave TEV protease. The protein stock was approximately 6.6 mg / mL in PBS and contained approximately 20% FGF21 protein truncated at residue 84. 5 μl of FGF21 stock was mixed with 45 μl 200 mM sodium acetate buffer (pH 5.0) and 0.5 μl of 100 mM TU3205-044 (2-AAP-PEG8, see Example 20) stock solution. The final molar ratio was 1 mM 2-AAP-PEG8 to approximately 30 μM FGF21. The reaction was allowed to proceed for 16 hours at room temperature and allowed to complete.

35A shows the mass spectrum of unreacted FGF21 and FIG. 35B shows the mass spectrum of the reaction mixture: PEGylation reaction proceeded to completion, resulting in a protein of 21792.4 Da which was 556 Da larger than the mass of unreacted material. This mass difference is expected for the 2-AAP-PEG8 modified FGF21.

Various FGF21 PCL mutants (expressed and purified as in Example 10) were PEGylated with 23 kDa 2-AAP-PEG (TU3205-052, see Example 37-3). Typically the protein concentration was between 100 and 400 μM and 23 kDa 2-AAP-PEG was added at a final concentration of 1 mM in PBS buffer (pH 7.4). The reaction was incubated at 4 ° C. for 3 days. SDS-PAGE of PEGylation with seven representative FGF21 PCL mutants is shown in FIG. 36A, and eight purified PEGylated FGF21 proteins isolated from full-length (FL) unreacted FGF21 and truncated (TR) FGF21. Is shown in Figure 36B.

Example 21 Derivatization of 2-amino-acetophenone- PEG of PCL incorporated into EPO

PCL was incorporated at various locations in mouse EPO as described in Example 6. After Ni-NTA purification, mEPO constructs (# 6, # 9 and # 10 mutant constructs) were further purified by S-300 gel filtration column in PBS. Purified mEPO protein was concentrated to approximately 1 mg / ml. Activated 23 kDa 2-AAP-PEG (TU3205-052, see Example 37-3) was added to the purified protein at 1 mM and incubated under the conditions shown in Table 6.

Figure pat00094

All mEPO constructs were run as monomers in the column. When 23 kDa 2-AAP-PEG was incubated with the purified protein, mEPO was PEGylated at a single site as it migrated to the 65 kDa band of SDS-PAGE (FIG. 37). The efficiency of the reaction varied from 10% to 15% depending on the conditions and PEGylated to a higher degree at pH 5.5 than at pH 7 and pH 8.5. In addition, temperatures above 4 ° C. did not significantly increase the degree of PEGylation.

Derivatization of the PCL only D- mixed in TE by industrial Min - hRBP and FAS: Example 22.

This example shows that the amino sugar, D-mannosamine, is directly coupled to PCL incorporated into two target proteins. This example presents a general scheme for glycosylation of PCL containing proteins (FIG. 38).

Human RBP4 Phe122PCL (mutant # 6) was prepared in HEK293F cells as in Example 2. 10 μl of 170 μM protein stock was added to 89 μl 10 × PBS buffer (pH 7.5) and mixed with 1 μl of 1 M D-mannosamine. hRBP4 Phe122PCL protein (17 μM) was incubated with 10 mM D-mannosamine at room temperature for 14 hours (no reaction was observed) and then incubated at 37 ° C. for 48 hours. 39 shows the mass spectrum of the reaction mixture after the 37 ° C. incubation period. In addition to the expected peak of unreacted hRBP4 at 23165.6 Da (expected 23166 Da), an estimated allocation peak for the D-mannosamine adduct was observed at 23300.0 Da. The mass increase of 164.4 Da, although close, was not equal to the expected increase of 161.1 Da for the putative reaction product shown in FIG. 38. In addition, a portion of the protein degraded into species was detected at 21007.8 Da.

In a second example, FAS-TE (11 μM) modified with PCL at position 2222, expressed in Escherichia coli and purified as in Example 8, was combined with 10 mM D-mannosamine for 72 hours at room temperature. Incubated at. The mass spectrum of the unreacted sample is shown in FIG. 40A, which contained the expected signal at 33250.4 Da corresponding to the unreacted protein, and the peak at 33360.4 Da is due to protein contamination in the sample. Mass spectrometry of the reaction mixture showed the expected signal at 33250.4 Da, corresponding to unreacted protein (FIG. 40B). An additional peak was found at 33408.8 Da, which is 158.4 Da larger than the starting material, which is assumed to be the peak of the reaction product shown in FIG. 37. The comparison of the two samples suggests that FAS-TE Leu2222PCL was converted to approximately 50% yield with D-mannosamine adduct under the reaction conditions.

Example 23: PCL-mediated cross-linking of FGF21 shares

This example shows that proteins can be covalently dimerized via bifunctional PCL-specific cross linkers. FGF21 incorporating PCL at position lysine 84 was expressed in Escherichia coli, refolded and purified as described in Example 10 except that the protein did not cleave TEV protease. FGF21 Lys84PCL was derivatized with cross linker TU633-010 (FIG. 43A; see Example 37-4 for synthesis). The protein stock was approximately 6.6 mg / mL in PBS and contained approximately 20% FGF21 protein cleaved at residue 84. 5 μl of FGF21 stock was mixed with 45 μl 200 mM sodium acetate buffer (pH 5.0). 0.1 μl of 5 mM stock solution of cross linker TU633-010 (in DMSO) was added at a final concentration of 10 μM cross linker and approximately 30 μM FGF21. Similarly, stock solution of FGF21 (pH 7.4) in 50 μl of PBS was reacted with 1 μl of 5 mM TU633-010 at 100 μM cross linker and FGF21 concentration of approximately 300 μM. FGF21 samples diluted with 200 mM sodium acetate buffer were prepared as controls and treated identically. After 16 hours at room temperature, aliquots of reaction and control samples were analyzed by mass spectrometry and SDS-PAGE analysis without purification. Mass spectra obtained for a pH 5.0 sample (FIG. 43B) show the expected mass of covalent dimer (43037.2 Da; 53% relative intensity of all FGF21 peaks), the expected mass of FGF21 with crosslinker attached at one end (21820.0 Da; 33 %) And unreacted FGF21 (21235.2 Da; 14%) clearly showed peaks. For pH 7.4 samples no covalent dimers were detected; 28% of FGF21 reacted monocross with the cross linker, whereas most proteins did not react (data not shown).

Adjustment of the reactants to protein concentration further increased the yield of covalent dimers. Specifically, 10 μl of FGF21 stock solution was mixed with 90 μl of 200 mM sodium acetate buffer (pH 5.0). 0.3 μl of 5 mM stock solution of cross linker TU633-010 (in DMSO) was added at a final concentration of 15 μM cross linker and approximately 30 μM FGF21. One sample was incubated for 4 days at room temperature and the second sample was incubated at 4 ° C. Samples in 10 × PBS (pH 7.5) were also prepared and incubated identically. For pH 5.0 samples incubated at room temperature, the peak of the covalent FGF21 dimer at the expected mass of 43034.4 Da was the predominant species, and unreacted FGF21 was not detected at 21233.6 Da. Some modified FGF21 was detected at 21818.4 Da with cross linker attached at one end (FIG. 44A). The reaction did not progress to the same extent as at pH 5.0 and 4 ° C., leaving approximately 19% of FGF21 unreacted; 40% was modified with a cross linker attached to one end, while a mass spectral peak intensity of approximately 41% was that of a covalent dimer. The reaction at pH 7.5 did not produce any covalent dimers represented by SDS-PAGE (FIG. 44B).

Site specific modification of pyrroline - carboxy -lysine ( PCL ) containing proteins by various other molecules .

In another embodiment, the coupling of a 2-aminobenzaldehyde (ABA) conjugate and a 2-aminoacetophenone (AAP) conjugate provided herein to a PCL-containing protein is in 10 × phosphate buffered saline (PBS), pH 7.0. It performed at 25 degreeC. The conjugation reaction was initiated by the addition of 10 μM PCL-containing protein and 100 μM ABA / AAP conjugate. Complete formation of the protein conjugate was confirmed by electrospray ionization-mass spectrometry (ESI-MS) or matrix assisted laser desorption / ionization (MALDI). Coupling of ABA / AAP DNA conjugates was analyzed by gel transfer assay using NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA). After quantitative coupling, the protein conjugate is dialyzed with 10 mM sodium phosphate buffer (pH 7.5) and the Amicon Ultra-4 centrifugal filter unit (Millipore Corporation) with a cutoff of 10 kDa. , Bedford, Mass.), And concentrated to 100 μM. Freshly prepared 200 mM NaCNBH 3 solution (dissolved in 10 mM phosphate buffer, pH 7.5) was then added to a final concentration of 20 mM. After allowing the reduction reaction to proceed at 25 ° C. for 2-4 hours, the reaction was quenched by the addition of 6 volumes of 10 mM sodium phosphate buffer (pH 7.5). The reduced protein conjugates were last buffer exchanged into the desired buffer using a NAP-5 column or PD10 column (GE Healthcare, Piscataway, NJ). Examples of non-limiting coupling of such 2-aminobenzaldehyde (ABA) conjugates and 2-aminoacetophenone (AAP) conjugates to various proteins are provided below.

Example 24 Coupling of Biotin Reagents

To confirm that biotin can be coupled to a protein having one or more PCL residues incorporated therein, use aBA-biotin reagent (X3626-140, Example 40) to synthesize mEGF-Tyr10PCL (see Example 12). Conjugated with. Coupling of biotin was followed by addition of 500 μM ABA-Biotin to 10 μM mEGF-Tyr10PCL in phosphate buffered saline (PBS, pH 7.0) and 0.5% (v / v) DMSO and reaction at 25 ° C. for 16 hours. Was carried out. Complete formation of the biotin conjugate was confirmed by ESI-MS (FIG. 47A; expected mass for unreacted protein = 7296; expected mass for coupled protein = 7902).

After quantitative coupling, freshly prepared 200 mM NaCNBH 3 solution (dissolved in PBS, pH 7.0) was added to a final concentration of 20 mM. After allowing the reduction reaction to proceed at 25 ° C. for 3 hours, the reaction was quenched by the addition of 6 volumes of PBS (pH 7.0). Excess NaCNBH 3 was removed by dialysis against PBS (pH 7.0) at 4 ° C. using a slide-A-Riser dialysis cassette (3,500 Da molecular weight cutoff, Pierce). The reduced biotin conjugates were concentrated using Amicon Ultra-4 centrifugal filter units (Millipore Corporation) with a cutoff of 3.5 kDa. After electrophoresis through a NuPAGE 4-12% bis-tris gel (Invitrogen), the biotinylated protein was transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer system (Invitrogen). Moved to. Biotin conjugates were then detected with horseradish peroxidase (HRP) conjugated to goat anti-biotin antibody (1: 100 dilution, Cell Signaling Technologies) and hyperfilm ECL (GE) Healthcare)) (FIG. 47B). Uncoupled mEGF-Tyr10PCL and fluorescein-conjugated mEGF-Tyr10PCL served as negative controls. Lane 1, 20 pmol mEGF-Tyr10PCL-ABA-biotin conjugate; Lane 2, 8 pmol mEGF-Tyr10PCL-ABA-biotin conjugate; Lane 3, 2 nmol mEGF-Y10PCL; Lane 4, 20 pmol mEGF-Tyr10PCL-ABA-fluorescein conjugate.

Example 25 Fluorescent Molecular Coupling

To confirm that the fluorescent molecule can be coupled to a protein having one or more PCL residues incorporated therein, mEGF-Tyr10PCL (Example 12) using an ABA-fluorescein reagent (3793-050, see Example 42) Fluorescein). Coupling of Fluorescein was performed by adding 1 mM of ABA-Fluorescein to 10 μM mEGF-Tyr10PCL in phosphate buffered saline (PBS, pH 7.0) and 0.5% (v / v) DMSO and 25 ° C. Reaction was carried out for 16 hours. Formation of fluorescein conjugates was confirmed by ESI-MS (FIG. 47C; expected mass for uncoupled protein = 7296; expected mass for coupled protein = 8062).

Fluorescein conjugates were reduced with 20 mM NaCNBH 3 at 25 ° C. for 3 hours. After quenching the reduction reaction with 6 volumes of PBS (pH 7.0), residual NaCNBH 3 was removed by dialysis against PBS (pH 7.0) at 4 ° C. using a slide-A-Riser dialysis cassette (3,500 Da molecular weight cutoff). . The conjugate was then concentrated to 1 μM using an Amicon Ultra-4 centrifugal filter unit with a cutoff of 3.5 kDa. Absorbance spectra in the 350-700 nm range of 1 μM mEGF-Tyr10PCL-ABA-fluorescein and 10 μM ABA-fluorescein using SpectraMax Plus (Molecular Devices) Obtained. They both exhibited maximum absorption at 500 nm.

Subsequently, the fluorescence spectra were read on a Spectramax Gemini fluorometer (Molecular Devices). The emission spectra for 1 μM mEGF-Tyr10PCL-ABA-fluorescein and 10 μM ABA-fluorescein were scanned at a step size of 2 nm from 510 nm to 750 nm while maintaining the excitation wavelength at 490 nm. Obtained by They both exhibited maximum emission at 522 nm. In addition, excitation spectra for 1 μM mEGF-Tyr10PCL-ABA-fluorescein and 10 μM ABA-fluorescein maintain the emission wavelength at 522 nm while the excitation wavelength is 300 nm to 510 nm in step size of 2 nm. Obtained by scanning.

Example 26 Polysaccharide Coupling

To confirm that the polysaccharide can be coupled to a protein in which one or more PCL residues are incorporated therein, mEGF-Tyr10PCL (implemented using ABA-disaccharide (3793-050; Example 42, MW 546.52) Example 12) was conjugated with disaccharides. Coupling reactions were performed by adding 1 mM ABA-disaccharide to 10 μM mEGF-Tyr10PCL mutant protein in PBS and 1% (v / v) DMSO, pH 7.0. The reaction was allowed to proceed for 16 hours at room temperature and analyzed by ESI-MS (FIG. 47D). Mass spectra showed the main peak at the expected mass (7825 Da) for the conjugated protein. The mass of uncoupled protein was 7296 Da.

Example 27 Immunomodulators: Mono- Nitrophenyl Coupling of hapten junction

To determine whether an immunomodulator can be coupled to a protein incorporated with one or more PCL residues, mTNF-Gln21PCL using ABA-mono-nitrophenyl hapten reagent (3793-001, Example 38-8) and mEGF-Tyr10PCL (see Examples 11 and 12) was conjugated with mono-nitrophenyl hapten. Coupling of the mono-nitrophenyl hapten conjugate was performed as described above. 48A is the ESI mass spectrum of 3793-001 conjugated to mTNF-Gln21PCL (expected mass for uncoupled protein = 19275; expected mass for coupled protein = 19614) and FIG. 48B shows mEGF-Tyr10PCL ESI mass spectrum of 3793-001 conjugated to (Expected mass for uncoupled protein = 7296; Expected mass for coupled protein = 7635).

Example 28 Immunomodulators: Di- Nitrophenyl Coupling of hapten junction

To determine whether an immunomodulator can be coupled to a protein incorporated with one or more PCL residues, mTNF-Gln21PCL and mEGF-Tyr10PCL using di-nitrophenyl hapten (TU3627-088, Example 38-7) (See Examples 11 and 12) were conjugated with di-nitrophenyl hapten. Coupling of the di-nitrophenyl hapten conjugate was performed as described above. 48C is the ESI mass spectrum of TU3627-088 conjugated to mTNF-Gln21PCL (expected mass for uncoupled protein = 19275; expected mass for coupled protein = 19688), and FIG. 48D is mEGF-Tyr10PCL ESI mass spectrum of TU3627-088 conjugated to (Expected mass for uncoupled protein = 7296; Expected mass for coupled protein = 7709).

Example 29 Immune Modulators: Coupling of TLR7 Agonists

To confirm that an immunomodulator can be coupled to a protein incorporated with one or more PCL residues, mEGF-Y10PCL (Example 12-3) using an ABATLR7 agonist reagent (X3678-114; see Example 38-3) ) Is conjugated with a TLR7 agonist. Coupling reactions were performed by adding 100 μM ABA-TLR7 agonist to 10 μM mEGF-Y10PCL mutant protein in 200 mM sodium acetate buffer and 1% (v / v) DMSO, pH 4.5. The reaction was allowed to proceed for 16 hours at room temperature and analyzed by ESI-MS (FIG. 49A). The mass spectrum showed the major peak at the expected mass (8763 Da) for the conjugated protein, and the mass of uncoupled protein was 7296 Da.

Example 30 Immune Modulators: Coupling of PADRE Peptides

MTNF-Gln21PCL and mEGF-Tyr10PCL (see Examples 11 and 12) were conjugated with PADRE peptides to confirm that immunomodulators and peptides could be coupled to a protein having one or more PCL residues incorporated therein. Coupling of the PADRE peptides was performed as described in the paragraph immediately following the subtitle “Site-Specific Modification of Pyrroline-Carboxy-Lysine (PCL) Containing Proteins with Various Other Molecules”. FIG. 50A is a MALDI-TOF mass spectrometric analysis of mTNF-Gln21PCL conjugated with PX2-PADRE (3465-143; see Example 38-11) at pH 5.0, and FIG. 50B is mTNF conjugated with PX2-PADRE at pH 7.5. MALDI-TOF mass spectrometric analysis of -Q21PCL. The expected mass for uncoupled protein is 19275 Da and the expected mass for coupled protein is 20842 Da. 50C is an ESI mass spectrometric analysis of mTNF-Gln21PCL conjugated with BHA-exPADRE (3647-104; see Example 38-10). The expected mass for uncoupled protein is then 19275 Da, and the expected mass for coupled protein is 21317 Da. In addition, FIG. 51 is an ESI mass spectrum showing coupling of BHA-exPADRE to mEGF-Tyr10PCL (expected mass 7296 Da for uncoupled protein; expected mass 9338 Da for coupled protein).

Example 31 Immunomodulators: Coupling of Phospholipids

To confirm that an immunomodulator and phospholipid can be coupled to a protein in which one or more PCL residues are incorporated therein, mEGF-Tyr10PCL (Example 12-1) using ABA phospholipid reagent (TU3627-092; Example 43-1) Phospholipids (DOPE). Coupling of ABA-DOPE to mEGF-Tyr10PCL (MW = 7296 Da) was performed for 16 h at 25 ° C. in 20 mM HEPES (pH 7.0) and 1% (v / v) DMSO. The conjugation reaction was initiated by adding 10 μM mEGF-Tyr10PCL and 100 μM ABA-DOPE. Formation of protein conjugates was confirmed by electrospray ionization-mass spectrometry (ESI-MS) analysis of DOPE conjugation to mEGF-Y10PCL (FIG. 49B; expected mass for uncoupled protein = 7296; conjugation Expected mass for protein isolated = 8227 Da).

Example 32 Protein : Oligonucleotide Coupling to CpG Peptides

To confirm that oligonucleotides and CpG immune modulators can be coupled to a protein having one or more PCL residues incorporated therein, CpG reagent BHA-BG1 (3647-057; see Examples 38-12) or CpG reagent BHA- MTNF-Gln21PCL and mEGF-Tyr10PCL (see Examples 11 and 12) were conjugated with CpG oligonucleotides using BG2 (3597-167; see Examples 38-14). Coupling of CpG oligonucleotides was performed as described in the paragraph immediately following the subtitle “Site-Specific Modification of Pyrroline-Carboxy-Lysine (PCL) -Containing Proteins by Various Other Molecules”. Coupling of BHA-BG1 (7.4 kDa) and BHA-BG2 (7.4 kDa) to mTNF-Gln21PCL (19.3 kDa) was confirmed by gel transfer assay (FIG. 52A) and BHA to mEGF-Tyr10PCL (7.2 kDa) Coupling of -BG2 (7.4 kDa) was also confirmed by gel transfer assay (FIG. 52B).

Example  33: Synthesis of reactive pyrrolysine analogs

Example 33-1: (S) -2-amino-6- (3- oxobutanamido ) hexanoic acid Hydrochloride Synthesis of (TU3000-016)

Figure pat00095

Bing Hao et al., ChemBio 2004, 11, 1317-24 & according to the procedure described in the data, wherein the removal of the Cbz group is carried out in the presence of HCl so that an HCl salt is obtained (S) -methyl 6-amino- 2- (2,2,2-trifluoroacetamido) hexanoate hydrochloride (TU2982-126) was prepared.

(S) -methyl 6-amino-2- (2,2,2-trifluoroacetamido) hexanoate hydrochloride (0.943 g, 3.22 mmol), N, N-diisopropyl in 40 mL glass vial To ethylamine (DIEA, 1.39 mL) and dichloromethane (DCM, 10 mL) was added diketone (0.37 mL) under N 2 atmosphere and the reaction was stirred at ambient temperature for 16 hours. The reaction mixture is diluted with ethyl acetate (EtOA), washed successively with H 2 O, 1 N HCl, H 2 O, saturated aq Na 2 CO 3 , and saturated aq NaCl, dried over Na 2 SO 4 , filtered And concentrated under reduced pressure. The residue was purified by SiO 2 flash chromatography to give (S) -methyl-6- (3-oxobutanamido) -2- (2,2,2-trifluoroacetamido) hexanoate ( TU3000-012) was obtained as a yellow oil.

Figure pat00096

(S) -methyl 6- (3-oxobutaneamido) -2- (2,2,2-trifluoroacetamido) hexanoate (0.736 g, 2.16 mmol) was dissolved in 1 N aq NaOH (6.5 mL). ) And 10 mL H 2 O at ambient temperature for 18 hours. LC-MS analysis was used to confirm the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was treated with excess 1 N aq HCl and concentrated to dryness under reduced pressure to afford (S) -2-amino-6- (3-oxobutanamido) hexanoic acid hydrochloride (TU3000-016) It was.

Figure pat00097

Synthesis of (S) -5- (4- acetyl-benz amido) - 1-carboxy-pentane-1-aminium chloride fried (TU3000-004): Example 33-2

Figure pat00098

(S) -Methyl 6-amino-2- (2,2,2-trifluoroacetamido) hexanoate hydrochloride (TU2982-126) (303 mg, 1.03 mmol), 4-acetylbenzoic acid (190 mg ), HATU (418 mg), DIEA (523 μl) and DMF (8 mL) were combined and stirred at ambient temperature for 18 hours. The reaction mixture is diluted with EtOA and washed successively with H 2 O, 1 N HCl, H 2 O, saturated aq Na 2 CO 3 , and saturated aq NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. I was. The residue was purified by SiO 2 flash chromatography to afford (S) -methyl-6-amino-2- (2,2,2-trifluoroacetamido) hexanoate hydrochloride (TU2982-136). Obtained.

Figure pat00099

(S) -Methyl 6- (4-acetylbenzamido) -2- (2,2,2-trifluoroacetamido) hexanoate (TU2982-136) (0.473 g) in 1 mL of 10 mL MeOH Treated with N aq NaOH (2.36 mL) at ambient temperature for 18 hours. LC-MS analysis confirmed that the hydrolysis of the methyl ester was complete and the trifluoroamide residues remained intact. The reaction was heated at 60 ° C. for 5 hours, at which point it was confirmed by LC-MS analysis that the amide hydrolysis was nearly complete. The reaction mixture was cooled down and concentrated under reduced pressure. The residue was treated with excess 1 N aq HCl and concentrated to dryness under reduced pressure to afford ((S) -5- (4-acetylbenzamido) -1-carboxypentane-1-aluminum chloride (TU3000- 004) was obtained as a pale yellow solid.

Figure pat00100

Example 33-3: ((S) -5- (5- Acetylthiophene- 2 -carboxamido ) -1- carboxypentane -1 -aluminum chloride (TU3000-006), (S) -5- (3-acetylbenzamido) -1-carboxypentane-1-aluminum chloride (TU3000-008), and (S) -5- (4-acetyl-1-methyl-1H-pyrrole-2-carboxamid Figure 1) Synthesis of 1-carboxypentane-1-aluminum chloride (TU3000-010)

Figure pat00101

(S) -5- (5-acetylthiophene-2-carboxamido) -1-carboxypentane-1-amid in the same manner as TU3000-004 except that the corresponding acid is used instead of 4-acetylbenzoic acid Chloride (TU3000-006), (S) -5- (3-acetylbenzamido) -1-carboxypentane-1-aminium chloride (TU3000-008), and (S) -5- (4-acetyl -1-methyl-1H-pyrrole-2-carboxamido) -1-carboxypentane-1-aluminum chloride (TU3000-010) was prepared. Acids used were 4-acetyl-1-methyl-1H-pyrrole-2-carboxylic acid, 5-acetylthiophene-2-carboxylic acid and 3- in the case of TU3000-006, TU3000-008, and TU3000-010, respectively. It was acetylbenzoic acid.

Figure pat00102

Example 33-4: (S) -2-amino-6- (3- oxocyclobutanecarboxamido ) hexanoic acid Synthesis of (TU3205-030).

Figure pat00103

Iodomethane (2.0 mL), K 2 CO 3 (5.60 g), (S) -6- (benzyloxycarbonylamino) -2- (tert-butoxycarbonylamino) hexanoic acid (1) (Novabio NovaBiochem, A29340), and anhydrous DMF (20 mL) were combined and stirred at ambient temperature for 2 hours. The reaction mixture was subjected to aqueous work up to afford (S) -methyl 6- (benzyloxycarbonylamino) -2- (tert-butoxycarbonylamino) hexanoate (TU3000-090) as a clear oil.

Figure pat00104

(S) -methyl 6- (benzyloxycarbonylamino) -2- (tert-butoxycarbonylamino) hexanoate (8.60 g) was dissolved in 150 mL MeOH on palladium (1.08 g) on 5% activated carbon. Hydrogenation at ambient temperature under atm for 3 hours. The spent catalyst was removed by vacuum filtration through a pad of celite and washed with MeOH. The combined filtrate and washings were concentrated under reduced pressure to afford (S) -methyl 6-amino-2- (tert-butoxycarbonylamino) hexanoate (TU3000-128) as a clear viscous oil.

Figure pat00105

A 1 M stock solution of (S) -methyl 6-amino-2- (tert-butoxycarbonylamino) hexanoate in 20 mL DMF was prepared and used. 2 mL aliquots of (S) -methyl 6-amino-2- (tert-butoxycarbonylamino) hexanoate stock solution, 3-oxocyclobutanecarboxylic acid (2) (Parkway * BX -102, 283 mg), HATU (800 mg), DIEA (1.0 mL) and 8 mL DMF were combined in 40 mL glass vials and stirred at ambient temperature overnight. LC-MS analysis confirmed the reaction was complete. The reaction mixture is diluted with EtOAc, water, saturated aq. Continuous washing with NaCl, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (hexane / EtOAc) to give (S) -methyl 2- (tert-butoxycarbonylamino) -6- (3-oxocyclobutanecarboxamido) hexanoate (TU3000-140) was obtained as a clear viscous oil.

Figure pat00106

(S) -Methyl 2- (tert-butoxycarbonylamino) -6- (3-oxocyclobutanecarboxamido) hexanoate (0.501 g) in 20 mL 4 M HCl in 1,4-dioxane The furnace was treated at ambient temperature for 20 minutes and the solvent was removed under reduced pressure. The resulting viscous oil was taken up in 10 mL CH 3 CN and seeded in small amounts of the title compound crystals. The resulting crystals were collected by vacuum filtration, washed with CH 3 CN and dried under reduced pressure to afford (S) -methyl 2-amino-6- (3-oxocyclobutanecarboxamido) hexanoate (TU3205). -016) was obtained as a colorless solid.

Figure pat00107

(S) -Methyl 2-amino-6- (3-oxocyclobutanecarboxamido) hexanoate (0.297 g) and 18 mL H 2 O were placed in a 40 mL glass vial. To the resulting clear solution was added 2.2 mL 1 N NH 4 OH and the reaction was shaken at ambient temperature for 22 hours, at which point the reaction was confirmed to be complete by LCMS analysis. The reaction mixture was frozen and lyophilized to afford (S) -2-amino-6- (3-oxocyclobutanecarboxamido) hexanoic acid (TU3205-030) as colorless crystals.

Figure pat00108

Example  34: Synthesis of Reactive Pyrrolysine Intermediates

Example 34-1: Synthesis of lithium 2- (4-acetyl-3-aminophenoxy) acetate (TU3205 -042)

Figure pat00109

1- (4-hydroxy-2-nitrophenyl) ethanone (Carbocore, 181 mg, 1.00 mmol), ethyl 2-bromoacetate (183 mg, 1.10 mmol), potassium carbonate (138 mg. 1.00 mmol) and DMF (5 mL) were combined in 20 mL glass vials and stirred at 60 ° C. for 2 hours at which point LC-MS analysis showed the reaction was complete. The reaction mixture was diluted with water, extracted with EtOAc and washed with saturated NaCl aq. 1- (4-hydroxy-2-nitrophenyl) ethanone (0.802 g, 4.43 mmol), ethyl 2-bromoacetate (0.813 g, 4.87 mmol), potassium carbonate (0.612 g. 4.43 mmol) and DMF (25 the reaction was repeated and worked up in the same manner. The combined EtOAc extracts were dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure to afford ethyl 2- (4-acetyl-3-nitrophenoxy) acetate (TU3205-034) as a dark yellow oil.

Figure pat00110

Ethyl 2- (4-acetyl-3-nitrophenoxy) acetate (TU3205-034) (111 mg) in MeOH (5 mL) was added to 10% palladium on charcoal (11 mg) at ambient temperature under H 2 atmosphere. Hydrogenated. After 30 minutes, LC-MS analysis confirmed that the reaction was complete. The reaction was repeated with TU3205-034 (1.45 g), palladium on 10% charcoal (140 mg), and MeOH (80 mL). The two reaction mixtures were combined and the spent catalyst was removed through a pad of celite by filtration. The filtrate was concentrated under reduced pressure to afford ethyl 2- (4-acetyl-3-aminophenoxy) acetate (TU3205-036) as a dark yellow oil.

Figure pat00111

Ethyl 2- (4-acetyl-3-aminophenoxy) acetate (TU3205-036) (1.02 g) was dissolved in THF (17 mL) and with aqueous LiOH (1 M, 4.3 mL) at ambient temperature for 1 hour. Treated. LC-MS analysis showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give lithium 2- (4-acetyl-3-aminophenoxy) acetate (TU3205-042) as a yellowish solid.

Figure pat00112

Example 34-2: Synthesis of lithium 2- (3-amino-4-formyl-phenoxy) acetate (TU3627 -018).

Figure pat00113

Borontribromide (24 g) was added dropwise to 4-methoxy-2-nitrobenzaldehyde (Carbocore, CO-0119, 5.5 g) and 200 mL DCM in a 500 mL round bottom flask while cooling in an ice bath. The reaction was stirred at the same temperature for 30 minutes and then at ambient temperature for 3 hours. The reaction mixture was carefully poured into ice water and left to stand at ambient temperature for 3 days. The aqueous mixture is extracted with EtOAc and saturated aq. Washed with NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude material was purified by SiO 2 gel flash chromatography using a linear gradient of EtOAc in 20 → 60% hexanes (Rf. 0.32, EtOAc in 50% hexanes) to give TU3627-002 as orange crystals.

Figure pat00114

TU3627-002 (1.87 g), ethyl bromoacetate (Aldrich, 1.5 mL), potassium carbonate (2.32 g) and DMF were combined and stirred at ambient temperature for 18 hours. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated and saturated aq. Washed with NaCl, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude was purified by SiO 2 gel flash chromatography using a linear gradient of EtOAc in 5 → 35% hexanes (Rf. 0.32, EtOAc in 35% hexanes) to afford TU3627-008 as a dark yellow oil.

Figure pat00115

TU3627-008 was reduced using the method described in Merlic, Camerlic et al., J. Org. Chem., 1995, 60, 3365-69. Specifically, TU3627-008 (1.717 g), iron powder (3.79 g), EtOH (45 mL), H 2 O (11 mL) and concentrated HCl (180 μl) were combined and heated to reflux for 2 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was subjected to silica gel using a linear gradient of solvent B in 5 → 60% solvent A (solvent A: NEt 3 in 5% hexanes; solvent B: NEt 3 in 5% EtOAc) (Rf. 0.34, EtOAc in 35% hexanes). Purification by flash chromatography gave TU3627-014 as light yellow crystals.

Figure pat00116

TU3627-014 (0.608 g) in 5 mL THF was diluted with 1 M aq. A 2.72 mL aliquot of LiOH was treated at ambient temperature. After 20 minutes, LC-MS analysis confirmed the reaction was complete. The reaction mixture was concentrated to dryness under reduced pressure to give lithium 2- (3-amino-4-formylphenoxy) acetate (TU3627-018) as a yellow solid.

Figure pat00117

Example 34-3: lithium 4- (3-acetyl-4-aminophenoxy) butanoate Synthesis of (TU3627 -064).

Figure pat00118

A mixture of 4'-chloro-2'-nitroacetophenone (Bionet cat # 3W0333, 1.00 g), potassium acetate (4.91 g) and DMSO was heated at 170 ° C. for 20 minutes under microwave irradiation. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated and saturated aq. Washed with NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. Another reaction was carried out in the same manner and the crude products from both reactions were combined and purified by silica gel flash chromatography (EtOAc). The title compound was obtained as an orange solid.

Figure pat00119

A mixture of 4'-hydroxy-2'-nitroacetophenone (1.12 g), ethyl 4-bromobutanoate (1.33 g), potassium carbonate (0.94 g) and 4 mL DMF was stirred at 60 ° C. for 6.5 hours. It was. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated and saturated aq. Washed with NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude product was purified by silica gel flash chromatography (hexane / EtOAc) to afford the title compound 1 as yellow crystals.

Figure pat00120

TU633-148 (1.48 g) was reduced by the method described in Merrick's [CAMerlic et al., J. Org. Chem., 1995, 60, 3365-69]. TU3627-056 is obtained as a light yellow oil after silica gel flash chromatography using a linear gradient of 5 → 60% solvent B in solvent A (solvent A: NEt 3 in 5% hexanes; solvent B: NEt 3 in 5% EtOAc). It was.

Figure pat00121

TU3627-056 (0.50 g) in 3 mL THF was diluted with 1 M aq. 1.9 mL aliquots of LiOH were treated for 18 hours at ambient temperature. LC-MS analysis confirmed the reaction was complete. The reaction mixture was concentrated to dryness under reduced pressure to give lithium 4- (3-acetyl-4-aminophenoxy) butanoate (TU3627-064) as a yellow solid.

Figure pat00122

Example 34-4: lithium 4- (3-amino-4-formyl-phenoxy) butanoate: Synthesis of (TU3627 -074).

Figure pat00123

A mixture of 2-nitro-4-hydroxybenzaldehyde (2.85 g), ethyl 4-bromobutanoate (3.66 g), potassium carbonate (2.84 g) and DMF (20 mL) was stirred at ambient temperature for 50 hours. . The reaction mixture was partitioned between H 2 O and EtOAc. The organic layer was separated and washed with saturated NaHCO 3 aq. The combined aqueous layers were extracted with EtOAc. The combined organic layers were washed with soft citric acid, saturated NaCl aq, dried over MgSO 4 , filtered and concentrated. The residue was purified by silica gel flash chromatography using a linear gradient of EtOAc in 20 to 40% hexanes (Rf: 0.35, EtOAc in 35% hexanes) to afford the title compound as a yellow oil.

Figure pat00124

TU3627-062 (3.89 g) was reduced by the method described in Merrick's [CAMerlic et al., J. Org. Chem., 1995, 60, 3365-69], as described herein. TU3627-066 was obtained as a yellow solid after silica gel flash chromatography using a linear gradient of solvent B in 20-60% solvent A (solvent A: NEt 3 in 5% hexanes; solvent B: NEt 3 in 5% EtOAc). . Rf: 0.51, EtOAc in 50% hexanes.

Figure pat00125

TU3627-066 (1.00 g) in 6 mL THF was treated with 3.98 mL of 1 M aqueous LiOH at ambient temperature for 4 hours. Most of the solvent was removed under reduced pressure, and the resulting turbid mixture was diluted with double deionized water, frozen and lyophilized to yield lithium 4- (3-amino-4-formylphenoxy) butanoate (TU3627-). 074) was obtained as a pale yellow solid.

Figure pat00126

Example 34-5: Synthesis of lithium 3- (3-acetyl-4 -aminophenyl ) propanoate ( X3547-1 ) .

Figure pat00127

1- (2-amino-5-bromophenyl) ethanone (642 mg), Pd (OAc) 2 (33.7 mg), and P ( o tolyl) 3 (137 mg) in anhydrous DMF (10 mL) in a pressure tube. To the mixture was added methyl acrylate (351 μl) and TEA (1.4 mL). The mixture was flushed with N 2 for 3 minutes, then sealed and heated at 110 ° C. for 4 hours. The reaction mixture was cooled to ambient temperature and then partitioned between ethyl acetate and water. The aqueous layer was extracted once with ethyl acetate and the combined organic layers were washed with brine, dried (Na 2 SO 4 ), filtered and the solvent removed in vacuo. The crude residue was purified by silica gel flash chromatography (EtOAc / hexanes) to afford the product.

Figure pat00128

(E) -methyl 3- (3-acetyl-4-aminophenyl) acrylate (219 mg) was reduced with 5% Pd / C (21.9 mg) in MeOH at room temperature with hydrogen balloon, after filtration and concentration To give the product (quantitative). The product was used in the next step without further purification.

Figure pat00129

Methyl 3- (3-acetyl-4-aminophenyl) propanoate (221 mg) and 4 M LiOH (0.275 mL) were added to 3 mL THF / water (v / v = 3/1). After the reaction was completed the solvent was removed under reduced pressure to give lithium 3- (3-acetyl-4-aminophenyl) propanoate (X3547-1).

Figure pat00130

Example 34-6: Synthesis of Lithium 3- (4-acetyl-3 -aminophenyl ) propanoate ( X3547-8 ) .

Figure pat00131

CH 3 CN solution (10 mL) containing 4-bromo-3-nitrobenzaldehyde (1.150 g) while stirring a solution of ethyl triphenylphosphoranilidine acetate (1.742 g) dissolved in CH 3 CN (15 mL) Was added. The reaction mixture was refluxed overnight. After cooling the mixture, the solvent was removed under reduced pressure to give a crude solid. After silica gel flash column chromatography, the pure product X3471-146 was obtained as a white solid (hexane / EtOAc, 9: 1).

The product X3471-146 (632 mg) and PdCl 2 (PPh 3 ) 2 (148 mg) from this step were dissolved in DMF (5.0 mL) under N 2 in a Schlenk tube. Tributyl (1-ethoxyvinyl) stannan (711 μl) was added with stirring. The mixture was heated at 100 ° C. overnight. After cooling the mixture, the solvent was removed under reduced pressure, diluted with DCM and washed with water and brine. After removal of DCM, the residue was purified by silica gel flash column chromatography (EtOAc in 15% -25% hexanes) to afford compound X3471-154.

Compound X3471-154A was treated with 20 mL 1 N HCl at room temperature for 4 hours. The solvent was removed to afford (E) -ethyl 3- (4-acetyl-3-nitrophenyl) acrylate X3471-154B.

Figure pat00132

Compound X3471-154B (263 mg) was diluted with ethyl 3- (4-acetyl-3-aminophenyl) propanoate X3547-2 at 1 atm under hydrogen using 5% Pd / C (26 mg) in 5.0 mL MeOH. Reduced.

Figure pat00133

Compound X3547-2 was treated at room temperature using a mixture of 4 M LiOH (0.248 mL), THF (3.0 mL) and water (1.0 mL). After the reaction was completed, the reaction mixture was lyophilized to give lithium 3- (4-acetyl-3-aminophenyl) propanoate (X3547-8).

Figure pat00134

Example 34-7: Lithium 5- (3-amino-4-formylphenyl) pentanoate synthesis of (X3547 -30).

Figure pat00135

4-bromo-2-nitrobenzaldehyde (460 mg), Pd (PPh 3 ) 2 Cl 2 (70 mg), and CuI (38 mg), methyl pent-4-inoate (269 mg) in TEA (5.0 mL) ) Was stirred at room temperature until the reaction was complete. The crude residue was purified by silica gel flash chromatography (EtOAc in 15% hexanes) to afford methyl 5- (4-formyl-3-nitrophenyl) pent-4-inoate.

Figure pat00136

Methyl 5- (4-formyl-3-nitrophenyl) pent-4-inoate (522 mg) was reduced at room temperature with 5% Pd / C (53 mg) in MeOH with a hydrogen balloon. Filtration followed by concentration under reduced pressure yielded methyl 5- (3-amino-4-formylphenyl) pentanoate.

Figure pat00137

Methyl 5- (3-amino-4-formylphenyl) pentanoate (447 mg) was treated with LiOH (88 mg) in 8.0 mL THF / water (v / v = 3/1). After the reaction was completed, the solvent was removed to obtain lithium 5- (3-amino-4-formylphenyl) pentanoate (X3547-30).

Figure pat00138

Example 34-8: Synthesis of 3- (4-acetyl-3 -aminophenyl ) -2 -aminopropanoic acid ( X3179-96 ) .

Figure pat00139

Sodium hydride (60% in mineral oil, 1.74 g) was washed with hexanes and dispersed in DMF (12 ml). Diethyl acetamidomalonate (10.4 g) in DMF (30 ml) and 4- (bromomethyl) -1-fluoro-2-nitrobenzene (10.2 g) in DMF (10 ml) were added successively, The reaction mixture was stirred for 4 hours at room temperature, then the solvent was removed under reduced pressure. The residue was purified by silica gel flash chromatography (EtOAc in 10-20% DCM) to give diethyl 2-acetamido-2- (4-fluoro-3-nitrobenzyl) malonate.

Figure pat00140

DBU (9.0 mL) is added to diethyl 2-acetamido-2- (4-fluoro-3-nitrobenzyl) malonate (7.41 g) and nitroethane (6.0 mL) in EtOAc and the mixture is brought to room temperature. Stir for 16 hours. The reaction mixture was concentrated and the residue was dissolved in methanol (35 mL). Aqueous H 2 O 2 (30%, 10.2 mL) and 10% aqueous NaHCO 3 (10.2 mL) were added and the mixture was stirred at rt for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with 1N HCl, brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (EtOAc in 10-20% DCM) to give diethyl 2-acetamido-2- (4-acetyl-3-nitrobenzyl) malonate.

Figure pat00141

Diethyl 2-acetamido-2- (4-acetyl-3-nitrobenzyl) malonate (2.0 g) was dissolved in 10 mL 37% HCl, heated at 100 ° C. overnight and cooled. The resulting solid was collected by filtration to give 3- (4-acetyl-3-nitrophenyl) -2-aminopropanoic acid.

Figure pat00142

3- (4-acetyl-3-nitrophenyl) -2-aminopropanoic acid was reduced under 1 atm H 2 using Pd on 5% carbon in MeOH. Filtration followed by concentration under reduced pressure afforded 3- (4-acetyl-3-aminophenyl) -2-aminopropanoic acid (X3179-96).

Figure pat00143

Example  34-9: 1- (2-amino-5- Bromophenyl ) Ethanone  ( X1436 -132).

Figure pat00144

The flask filled with 1- (2-aminophenyl) ethanone (135 mg) and DCM (2.5 mL) was cooled to −10 ° C. and NBS (178 mg) was added in several portions over 30 minutes. The reaction mixture was diluted with 10 mL DCM, washed with saturated aqueous NaHCO 3 , dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to afford the title compound.

Figure pat00145

Example  34-10: 1- (2-amino-5- Iodophenyl ) Ethanone  ( X1436 -134).

Figure pat00146

The flask filled with 1- (2-aminophenyl) ethanone (405 mg) and DCM (20 mL) was cooled in an ice / water bath and NIS (675 mg) was added in three portions. The reaction was stirred at 0 ° C. The reaction mixture was diluted with 30 mL DCM, washed with saturated aqueous NaHCO 3 , dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude material was purified by preparative RP-HPLC to afford the title compound.

Figure pat00147

Example  34-11: 2-amino-4- Bromobenzaldehyde  ( X3547 -158).

Figure pat00148

Iron powder (1.20 g), water (2.0 mL), and HCl (12 N, 40 μl) were added sequentially to a solution of 2-nitrobenzaldehyde (230 mg) in ethanol (8.0 mL). After stirring at 95 ° C. for 90 minutes, the reaction mixture was filtered to high temperature. After washing with ethanol, the filtrates were combined and the solvent removed in vacuo. The crude material was purified by silica gel flash chromatography (40: 55: 5 hexanedecyl acetate / triethylamine) to give 2-amino-4-bromobenzaldehyde as yellow crystals.

Figure pat00149

Example  35: PCL  And synthesis of pyrrolysine biosynthetic precursors

Example  35-1: ammonium DL -One- Pyrroline -5- Carboxylate  Synthesis of (3793-007).

Figure pat00150

HCl salt of H-DL-δ-DL-hydroxyLys-OH (118 mg, 0.5 mmol) was applied to a cation exchange SPE cartridge and the amino acid was eluted with ammonium hydroxide. NaIO 4 (107 mg) was added to the ammonium eluate and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was lyophilized and the crude product was acidified to pH 2.6 with HCl and purified by cation exchange SPE. After decanting the first 10 mL of H 2 O eluate, the next 30 ml of H 2 O eluate was collected and immediately neutralized with 1 N NH 4 OH ( aq ) (0.5 mL). After lyophilization, the desired product was obtained as a light yellow powder.

Figure pat00151

Example  35-2: H-L- Lys -N ε -(D- Orn ) - OH  Synthesis of (3793-031)

Figure pat00152

Boc-D-Orn (Boc) -OH (1.097 g) was treated with HATU (1.255 g) and DIEA (1.53 mL) in DMF (5 mL) for 1 hour. Then DMF solution (5 mL) and DIEA (627 μL) of Boc-Lys-OMe (acetate salt, 1.057 g) were added to the reaction mixture and the reaction stirred at room temperature for 2 hours. The reaction mixture was partitioned between 10% NaCl ( aq ) (30 mL) and EtOAc (60 mL). The EtOAc layer was washed with 5% citric acid (30 mL), 10% NaHCO 3 (aq) (30 mL), and brine (30 mL). EtOAc layer was dried over Na 2 S0 4 (s) , filtered and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography using 80 g silica column and gradient elution of 0-15% MeOH / DCM, Boc-Lys (Boc-D-Orn (Boc))-OMe (3793-026) Was obtained as a colorless oil.

Figure pat00153

To a 65% acetonitrile aqueous solution (5 mL) of Boc-Lys (Boc-D-Orn (Boc))-OMe (214 mg) was added concentrated HCl (1 mL) and the reaction mixture was stirred vigorously for 2 hours. The reaction mixture was lyophilized and purified by 1 N NH 4 OH in 20% MeCN ( aq ) by cation exchange chromatography using an SCX SPE cartridge. Methyl esters were hydrolyzed overnight in NH 4 OH eluent. After freeze-drying, HL-Lys-N ε - to give a (D-Orn) -OH (3793-031 ) as a white powder.

Figure pat00154

Example 35-3 N-((6 -chloropyridin- 3 - yl) methyl ) -1 -pyrroline -5 -carboxamide Synthesis of (3647-061).

Figure pat00155

T-butylpyrocarbonate (6.55 g) in dioxane (10 mL) was added dropwise to DL-δ-DL-hydroxyLys (1.99 g) in 1 N NaOH ( aq ) (50 mL) and the reaction was allowed to come to room temperature. Stir overnight at. The reaction mixture was acidified to pH 3.0 with 1 N HCl ( aq ) and extracted twice with EtOAc (200 mL). EtOAc layers were combined, dried over Na 2 SO 4 (s) , filtered and concentrated under reduced pressure to afford the crude product. The crude product was purified by silica gel flash column chromatography with a gradient elution of 0-10% MeOH / DCM to give Boc-DL-δ-DL-hydroxyLys (Boc) (3597-109) as a white solid.

Figure pat00156

Boc-DL-δ-DL-hydroxyLys (Boc) (435 mg) was treated with HATU (456 mg) and DIEA (523 μl) in 3.5 mL DMF for 1 hour. The resulting solution was added to 6-chloropyridin-3-ylmethylamine (143 mg) in 1.5 mL DMF solution at room temperature and the reaction stirred for 2 days. The reaction mixture was extracted with 10% NaCl ( aq ) (15 mL) and EtOAc (30 mL). The EtOAc layer was washed with 5% citric acid (15 mL), 10% NaHCO 3 (15 mL), and brine (15 mL). The EtOAc layer was then dried over Na 2 SO 4 (s) , filtered and concentrated under reduced pressure to afford the crude product. The crude product was purified by silica gel flash column chromatography with a gradient elution of 0-15% MeOH / DCM to afford the desired product (3647-016) as a white solid.

Figure pat00157

The above product (3647-016) (263 mg) was stirred in a solution of Et 2 O (7 mL) and 1 N HCl ( aq ) (7 mL) overnight at room temperature. After the reaction mixture was evaporated and lyophilized, the residue was purified by cation exchange SPE cartridge to afford the desired product (3647-037) as a white solid.

Figure pat00158

The above product (3647-037) (69.6 mg) was dissolved in 2 mL H 2 O (2 mL) and the pH was adjusted to 12 with 1 N NaOH (aq) . PL-IO 4 resin (Varian, 381 mg, 0.48 mmol) was added and the reaction stirred at rt for 2 h. The resin was removed by filtration and half of the filtrate was purified by preparative HPLC to give the desired product (3647-061) as a white solid.

Figure pat00159

Example  36: PCL  Synthesis of Model Compounds

Example  36-1: H-L- Lys -Nε- ( DL -One- Pyrroline -5-carbonyl)- OH  Synthesis of (3647-125).

Figure pat00160

Boc-DL-δ-DL-hydroxyLys (Boc) (3597-109) (886 mg) was activated for 1 hour by HATU (932 mg) in 3.0 mL DMF in the presence of DIEA (1068 μl), 3.0 To Boc-Lys-OMe (549 mg) in mL DMF (3.0 mL). The reaction was stirred at rt for 36 h. The reaction mixture was partitioned between 10% NaCl ( aq ) (30 mL) and EtOAc (60 mL). The EtOAc layer was washed with 5% citric acid (15 mL), 10% NaHCO 3 (15 mL), and brine (15 mL). EtOAc layer was dried over Na 2 S0 4 (s) , filtered and concentrated under reduced pressure to afford the crude product. The crude product was purified by silica gel flash column chromatography using 0-15% MeOH / DCM and then purified by RP-C18 SPE using elution of 25% and 65% MeCN ( aq ) to give the desired product (3647). -099) was obtained as a white solid.

Figure pat00161

To Boc-Lys (Boc-DL-δ-DL-hydroxyLys (Boc))-OMe (3647-099) (84.1 mg) in 1 mL Et 2 O was added 4 N HCl ( aq ) (3 mL) The reaction was stirred at rt for 3 h. Removal of the solvent gave 61.1 mg of crude product (3647-120) as a white solid.

Figure pat00162

The crude product (3647-120) (61.1 mg) was dissolved in 1 mL H 2 O and the pH was adjusted to 12 using 1 N NaOH (aq) . The mixture was stirred at rt for 1 h to hydrolyze the methyl ester. NaIO 4 (29.9 mg) was then added to the reaction mixture and the reaction was stirred for an additional 15 minutes at room temperature. The reaction mixture was neutralized with 1 N HCl (aq) and loaded into cation exchange SPE for purification, yielding HL-Lys-Nε- (DL-1-pyrroline-5-carbonyl) -OH (3647-125). Obtained.

Figure pat00163

Example  36-2: H-L- Lys -N ε - ( DL -One- Pyrroline 2-carbonyl)- OH  Synthesis of (3793-011).

Figure pat00164

Sodium 1-pyrroline-2-carboxylate (3647-164) was prepared using the following method described in Gyoergy Szoelloesi et al., Chirality, 2001, 13 (10), 619-624. .

Triethylamine (8.4 mL) was added to H-Pro-OMe HCl (7.512 g) in ether (27 mL) and the reaction stirred for 2 hours. The reaction mixture was filtered and Et 2 O was removed by evaporation. The residue was purified by vacuum distillation to give 4.119 g of H-Pro-OMe as a colorless oil. t-BuOCl (3.59 mL) was added dropwise to a solution of Et 2 O (100 mL) of H-Pro-OMe (4.089 g) and the reaction stirred at −50 ° C. for 1 h. The reaction mixture is allowed to warm to room temperature, triethylamine (4.64 mL) is added to the reaction mixture and then stirred for 3 days. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by vacuum distillation to afford methyl pyrroline-2-carboxylate (3647-154) as a colorless oil.

Figure pat00165

Methyl pyrroline-2-carboxylate (2.265 g) was dissolved in 1 N NaOH ( aq ) (17.8 mL) and stirred at room temperature for 1 hour. The reaction mixture was lyophilized to give crude sodium pyrroline-2-carboxylate (3647-164) as a white powder.

Figure pat00166

Sodium 1-pyrroline-2-carboxylate (459 mg) and DPPA (886 μl) are added to Boc-Lys-OMe (acetate salt, 897 mg) and DIEA (1184 μl) in 20 mL DMF and the reaction is added. Stir at room temperature under N 2 for 24 h. The reaction mixture was partitioned between 10% NaCl ( aq ) (50 mL) and EtOAc (100 mL). The EtOAc layer was dried over Na 2 S0 4 (s) , filtered and concentrated under reduced pressure to afford the crude product as a bright orange oil. The crude product was purified by silica gel flash column chromatography with a gradient elution of 0-15% MeOH / DCM and then by RP-C18 SPE to give the desired product (3647-167) as a colorless oil.

Figure pat00167

HCl (dark, 1 mL) was added to 50% MeCN (aq) of the product (3647-167) (49.3 mg). To the solution (5 mL) was added and the reaction stirred at rt for 2 h. After lyophilization, the residue was purified by cation exchange SPE cartridge. The methyl ester was hydrolyzed overnight with NH 4 OH ( aq ) in the eluent. After lyophilization, the desired product (3793-011) was obtained as a light yellow powder.

Figure pat00168

Example  37: 2- AAP - PEG  Synthesis of Reagents

The synthesis of other 2-AAP-PEG derivatives used in the examples provided herein is described below. 2-amino-acetophenone derivatives of PEG having molecular weights of approximately 500, 2400, 23000 Da as well as 5000 and 10000 Da were synthesized as follows:

Example  37-1: TU3205 -044 (0.5 kDa  2- AAP - mPEG ) Synthesis of

Figure pat00169

Lithium 2- (4-acetyl-3-aminophenoxy) acetate (TU3205-042) (55.9 mg) was charged to a 10 mL round bottom flask and DMF (3 mL) and HATU (98.9 mg) were added. The resulting slurry was stirred at ambient temperature for 40 minutes. During this time the reaction turned to a yellow solution. To the reaction was then added mPEG-amine (Quanta Biodesign, MW 383.5, 100 mg) in 2 mL DMF. LC-MS analysis after 5 minutes indicated the reaction was complete. The reaction mixture was stirred for a further 40 minutes and concentrated under reduced pressure. The residue was purified by SiO 2 flash chromatography (MeOH in 3% DCM) to give TU3205-044 (0.5 kDa 2-AAP-mPEG) as a yellow viscous oil.

Figure pat00170

Example  37-2: TU3205 -048 (2.4 kDa  2- AAP - PEG ) Synthesis of

Figure pat00171

Lithium 2- (4-acetyl-3-aminophenoxy) acetate (TU3205-042) (12.2 mg) was charged to a 10 mL round bottom flask and DMF (1 mL) and HATU (21.5 mg) were added. The resulting slurry was stirred at ambient temperature for 35 minutes. During this time the reaction turned to a yellow solution. To the reaction was then added mPEG-amine (Quanta Biodesign, MW 2209, 100 mg) in 2 mL DMF. The reaction mixture was stirred for 18 hours and concentrated under reduced pressure. The residue was purified by SiO 2 flash chromatography (MeOH in DCM) to give TU3205-048 (2.4 kDa 2-AAP-mPEG) as a yellow viscous oil.

Figure pat00172

Example  37-3: TU3205 -052 (23 kDa  2- AAP - PEG ) Synthesis of

Figure pat00173

Lithium 2- (4-acetyl-3-aminophenoxy) acetate (TU3205-042) (21.5 mg) and HATU (38.0 mg) were charged to a 10 mL round bottom flask and DMF (0.5 mL) was added. The resulting slurry was stirred at ambient temperature for 50 minutes. The resulting yellow solution was added to mPEG-NH 2 (Laysan Bio, average MW 23k, average n = 520, 0.50 g) in 5 mL DMF in 20 mL glass vial. The reaction was shaken for 2.5 hours at ambient temperature. The reaction mixture was then diluted with 10 mL water, 2.5 mL aliquots of each solution were applied to a PD-10 column (GE Healthtech) and the desired product was eluted with water according to the supplier's instructions. The pooled aqueous solution was pooled, frozen and lyophilized to give TU3205-052 (23 kDa 2-AAP-mPEG) as a white solid. (Note: PEG reagents characterized by high MW were obtained only after they were conjugated to PCL containing proteins.)

Figure pat00174
And

Figure pat00175

Was prepared in a similar manner to TU3205-052 using the corresponding 10,000 (mean n = 225) and 5,000 (mean n = 111) MW mPEG-NH 2 . (Note: PEG reagents characterized by high MW were obtained only after they were conjugated to PCL containing proteins.)

Example  37-4: TU633 -010: 2- Sensuality  Synthesis of Linkers

Figure pat00176

Lithium 2- (4-acetyl-3-aminophenoxy) acetate (TU3205-042) (94.6 mg) and HATU (167 mg) were charged to a 10 mL round bottom flask and DMF (2 mL) was added. The resulting slurry was stirred at ambient temperature for 45 minutes. To the resulting yellow solution was added 4,7,10-trioxa-1,13-tridecanediamine (Fluka, 44 mg) in 1 mL DMF. The reaction mixture was stirred at ambient temperature for 18 hours and concentrated under reduced pressure. The residue was purified by SiO 2 flash chromatography (MeOH in DCM) followed by preparative reverse phase LC purification. LC purified material was dissolved in EtOAc and treated with saturated aqueous NaHCO 3 to remove trifluoroacetic acid. The solvent was evaporated to give the bifunctional linker TU633-010 as a clear oil.

Figure pat00177

Example  37-5: m- PEG - AAP  29k ( TU633 -084).

Figure pat00178

Lithium 2- (4-acetyl-3-aminophenoxy) acetate (187 mg) and HBTU (330 mg) were placed in a 20 mL glass vial and 10 mL anhydrous DMF was added. The resulting slurry was stirred at ambient temperature. Within 20 minutes the reaction turned to a yellow solution and after 80 minutes a 9.5 mL aliquot of the reaction mixture was added to mPEG-NH 2 (Raysan Bio, MW 28,700) dissolved in 40 mL of anhydrous DMF in a 100 mL round bottom flask. It was. The reaction was shaken at ambient temperature for 19 hours. The reaction mixture was then applied to 24 pieces of PD-10 column (GE Helltech) and the desired product was eluted with water according to the supplier's instructions. The pooled eluate was frozen and lyophilized to give a white solid. The solid was dissolved in double deionized water and thoroughly dialyzed against double deionized water using a dialysis membrane of MWCO 3500. The dialyzed solution was frozen and lyophilized to give TU633-084 as a white downy solid. (Note: PEG reagents characterized by high MW were obtained only after they were conjugated to PCL containing proteins.)

Example  37-6: mPEG - AAP -30k ( TU633 Synthesis of -120)

Figure pat00179

Lithium 3- (3-acetyl-4-aminophenyl) propanoate (139 mg) and HBTU (247 mg) were placed in a 20 mL glass vial and 13 mL anhydrous DMF was added. The resulting slurry was stirred at ambient temperature for 30 minutes and the resulting solution was used to prepare TU633-120, TU633-122, TU633-124 and TU633-126. MPEGamine (NOF Corp., SUNBRIGHT MEPA-30T MW 30,298, 3.0 g) was placed in a 45 mL glass vial, 20 mL anhydrous DMF was added, and then lightly heated to dissolve the mPEGamine in DMF. To the resulting mPEGamine solution was added a 2.2 mL aliquot of the activated ester solution and the vial was shaken at ambient temperature for 18 hours and then at 37 ° C. for 24 hours. The reaction mixture was transferred to dialysis membrane tubing (Fisher Scientific, cat # 21-152-9, MWCO 3500) and thoroughly dialyzed over two days against double deionized water. The dialyzed solution was frozen and lyophilized to give TU633-120 as a white, cotton-like solid. (Note: PEG reagents characterized by high MW were obtained only after they were conjugated to PCL containing proteins.)

Instead of SUNBRIGHT MEPA-30T, NOF Corp. In a similar manner except using the corresponding activated PEG, SUNBRIGHTMEPA-40T (MW 42036), SUNBRIGHT GL2-400PA (MW 42348), and SUNBRIGHT GL4-400PA, respectively.

Figure pat00180

Figure pat00181
And

Figure pat00182
Was prepared.

(Note: PEG reagents characterized by high MW were obtained only after they were conjugated to PCL containing proteins.)

Example  37-7: mPEG - ABA -30 kD  ( TU3627 -024).

Figure pat00183

The title compound is the same as TU633-120 except that lithium 2- (3-amino-4-formylphenoxy) acetate is used instead of lithium 3- (3-acetyl-4-aminophenyl) propanoate Prepared. (Note; PEG reagents featuring high MW were obtained only after they were conjugated to PCL containing proteins.)

Example  37-8: mPEG - ABA -30k ( TU3627 -084).

Figure pat00184

The title compound is identical to TU633-120, except that lithium 4- (3-amino-4-formylphenoxy) butanoate is used instead of lithium 3- (3-acetyl-4-aminophenyl) propanoate. Prepared in the manner. H NMR analysis confirmed that there was no unmodified starting PEG present in the product. Endpoint Activity: greater than 95% by H-NMR.

Example  37-9: mPEG - ABA -40k ( TU3627 -086).

Figure pat00185

The title compound was prepared in the same manner as TU3627-084, except that SUNBRIGHTMEPA-40T was used instead of SUNBRIGHTMEPA-30T. H NMR analysis confirmed that there was no unmodified starting PEG present in the product. Endpoint Activity: greater than 95% by H-NMR.

Example 37-10: N 1 - (18- ( 3- amino-4-formyl-phenoxy) -15- oxo -4,7,10- tree-oxa-14-aza-octadecyl) -N 19 - (18 -(3-amino-4-formylphenoxy) -15-oxo-4,7,10-trioxa-9,14-diazatodecyl) -4,7,10,13,16-pentaoxanona Synthesis of Deccan-1,19-diamide (X3678-40)

Figure pat00186

Lithium 4- (3-amino-4-formylphenoxy) butanoate (94.5 mg) was activated with HBTU (151.7 mg) in 4 mL DMF. Diamido-dPEG ™ 11 -diamine (Quantabiodesign, Cat # 10361, 74.3 mg) was added to the reaction mixture and stirred at rt overnight. DIEA (17.5 μl, 0.1 mmol) was added and the mixture was heated at 40 ° C. for several hours. The title compound was isolated by preparative RP-HPLC and passed through a PL-HCO 3 MP SPE cartridge (Varian Inc.) to remove TFA.

Figure pat00187

Example  38: Immune Modulators to Proteins Coupling  Of reagents for

Example 38-1: N- (1- (3- amino-4-formyl-phenoxy) -2-oxo -7,10,13- tree oxa-3-aza-hexadecane -16- yl) -4- ((4- (2- (5-amino-8-methylbenzo [f] [1,7] naphthyridin-2-yl) ethyl) -3-methylphenoxy) methyl) benzamide (TU3627-042) synthesis.

Figure pat00188

T-butyl 3- (2- (2- (3-aminopropoxy) ethoxy) ethoxy) propylcarbamate (Quantabiodesign, cat # 10225, 500 mg) and DIEA (348 μL in 10 mL DCM ) Was added 4-chloromethylbenzoyl chloride in 2 mL DCM while cooling in an ice bath. The reaction was stirred at the same temperature for 1 hour. The reaction mixture is diluted with EtOAc, washed successively with H 2 O, light aqueous citric acid, aqueous NaHCO 3 and saturated aqueous NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the product (A) as clear Obtained as a viscous oil.

Figure pat00189

Product A (303 mg), Compound B (200 mg), cesium carbonate (208 mg) and anhydrous DMSO were combined and the reaction stirred at ambient temperature. After 18 hours, an additional 60 mg of Compound A and an additional 30 mg of cesium carbonate dissolved in 8 mL DMSO were added to the reaction and the reaction was stirred for 40 hours at ambient temperature, at which time Compound B only traced. Only amount remained (identified by LCMS analysis). The reaction mixture was diluted with EtOAc, washed successively with H 2 O, saturated aqueous NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography using a linear gradient of B (A: DMC; B: NH 3 in 2% MeOH) in 1 → 5% solvent A to afford the partially purified desired compound. After recrystallization from MTBE, the product was obtained as pale yellow crystals.

Figure pat00190

Boc groups of compound C were removed by treatment with 3 M methanolic HCl at ambient temperature. The reaction mixture was concentrated under reduced pressure to afford compound D as dihydrochloride salt.

Figure pat00191

Lithium 2- (3-amino-4-formylphenoxy) acetate (20 mg) and HBTU (38 mg) were placed in a 20 mL glass vial and 2 mL anhydrous DMF was added. The resulting slurry was stirred at ambient temperature for 30 minutes, then compound D (38 mg) and DIEA (52 μl) were added. The reaction was stirred at ambient temperature for 17 hours. LCMS analysis confirmed that no starting material remained, but a bis-acylated byproduct was formed with the desired product. The reaction mixture was diluted with EtOAc, washed successively with H 2 O and saturated aqueous NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was taken up in 5 mL THF and treated with 1 mL aliquots of 1 M aqueous LiOH for 2 hours at ambient temperature, at which point LC-MS analysis showed that the bis-acylated product was hydrolyzed to the desired product. It was shown. The reaction mixture was partitioned between EtOAc and water, the organic layer was separated, washed with saturated aqueous NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude material was subjected to preparative RP-HPLC for purification. The HPLC eluate containing the desired product was diluted with EtOA and washed with saturated aqueous NaHCO 3 and saturated aqueous NaCl to remove trifluoroacetic acid, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure, Compound N- (1- (3-amino-4-formylphenoxy) -2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl) -4-((4- ( 2- (5-amino-8-methylbenzo [f] [1,7] naphthyridin-2-yl) ethyl) -3-methylphenoxy) methyl) benzamide (TU3627-042) was obtained.

Figure pat00192

Synthesis of 4- (2- (5-amino-8- methylbenzo [f] [1,7] naphthyridin- 2 - yl) ethyl) -3 -methylphenol (compound B)

In a round bottom flask capped with a septum, 1-ethynyl-4-methoxy-2-methylbenzene (commercially available) (1.1 equiv), 3,5-dichloropicolininonitrile (1 equiv), triethylamine (5 equiv) , And anhydrous DMF (0.2 M) were added. Vacuum treatment and nitrogen flushing were performed three times. CuI (0.05 equiv) and bis (triphenylphosphine) dichloro-palladium (II) (0.05 equiv) were added. The diaphragm was replaced with a reflux condenser and the flask was heated at 60 ° C. overnight under nitrogen atmosphere. Upon completion of the reaction by monitoring by TLC, the contents of the flask were loaded onto a large silica gel column pretreated with hexane. Flash chromatography (silica gel, hexanes: EtOAc (1: 4%)) gave the product 3-chloro-5-((4-methoxy-2-methylphenyl) ethynyl) picolinonitrile.

Round bottom flask with reflux condenser in 3-chloro-5-((4-methoxy-2-methylphenyl) ethynyl) picolinonitrile (from previous step) (1 equiv), tert-butyl 2- (4 , 4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenylcarbamate (1.25 equiv), K 3 PO 4 (2 equiv), tris (dibenzylideneacetone) Dipalladium (0) (0.05 equiv), and 2-dicyclohexylphosphino-2 ', 6'-dimethoxybiphenyl (0.1 equiv) were added. n-butanol and water (5: 2, 0.2 M) were added and the contents were degassed three times (vacuum and nitrogen flushed). The reaction mixture was stirred vigorously overnight at 100 ° C. under nitrogen in an oil bath. The contents were cooled, dissolved in 200 mL of water and extracted with methylene chloride. The combined organic layers were dried (Na 2 SO 4 ) and concentrated. Product 2-((4-methoxy-2-methylphenyl) ethynyl) -8-methylbenzo [f] [1,7] naphth by flash chromatography (silica gel, EtOAc in 0-50% CH 2 Cl 2 ). Ridine-5-amine was obtained.

2-((4-methoxy-2-methylphenyl) ethynyl) -8-methylbenzo [f] [1,7] naphthyridin-5-amine (from the step above) with stirring in a round bottom flask ) (1 equivalent) was added. Ethanol and methylene chloride (1: 2, 0.2 M) were added followed by palladium in carbon (activated powder, wet, 10% on carbon, 0.1 equiv). The contents were evacuated and then hydrogen flushed three times. The reaction mixture was stirred vigorously overnight at room temperature under a hydrogen balloon. The reaction mixture was then filtered through a pad of celite and the pad of celite was subsequently washed with methylene chloride and EtOAc until no UV absorption occurred in the filtrate. The combined organic washes were concentrated. Product 2- (4-methoxy-2-methylphenethyl) -8-methylbenzo [f] [1,7] naphthyridine- by flash chromatography (silica gel, EtOAc in 0-50% CH 2 Cl 2 ). 5-amine was obtained.

Figure pat00193

CH 2 Cl to a stirred solution of 2- (4-methoxy-2-methylphenethyl) -8-methylbenzo [f] [1,7] naphthyridin-5-amine in methylene chloride in an ice-water bath (0.2 M) a 1 N solution of BBr 3 (2 eq) of 2 was added drop wise. Within 30 minutes the reaction was quenched with methanol and concentrated in vacuo to afford a crude residue. The crude material was purified by flash chromatography on a COMBIFLASH® system (ISCO) using 0-20% methanol in dichloromethane to give 4- (2- (5-amino-8-methylbenzo [f] [1,7] naphthyridin-2-yl) ethyl) -3-methylphenol was obtained as a white solid.

Figure pat00194

Example 38-2: 4-((4- (2- (5-amino-8- methylbenzo [f] [1,7] naphthyridin- 2 - yl) ethyl) -3-methylphenoxy) methyl Synthesis of) -N- (1- (aminooxy) -2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl) benzamide hydrochloride (TU3627-044).

Figure pat00195

HBTU (38 mg), 2- (tert-butoxycarbonylaminooxy) acetic acid (Fluka, 25 mg), DIEA (44 μl) and 5 mL DMF were combined in 20 mL glass vials. The reaction was stirred at ambient temperature for 30 minutes, then compound D (38 mg) was added. The reaction was stirred at ambient temperature for 17 hours. LC-MS analysis indicated no starting material remained, but several overacylated byproducts formed with the desired product. The reaction mixture was diluted with EtOAc, washed successively with H 2 O and saturated aqueous NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was taken up in 5 mL THF and treated with 1 mL aliquots of 1 M aqueous LiOH for 2 hours at ambient temperature, at which point LC-MS analysis showed that the overacylated product was hydrolyzed to the desired product. Indicated. The reaction mixture was partitioned between EtOAc and water, the organic layer was separated, washed with saturated aqueous NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography using B (A: DMC; B: NH 3 in 2% MeOH) in 5% solvent A to afford the desired compound, E.

Figure pat00196

Compound E (28 mg) was treated with 3 M methanolic HCl at ambient temperature for 30 minutes and concentrated under reduced pressure. This operation was repeated. Title compound 4-((4- (2- (5-amino-8-methylbenzo [f] [1,7] naphthyridin-2-yl) ethyl) -3-methylphenoxy) methyl) -N- ( 1- (aminooxy) -2-oxo-7,10,13-trioxa-3-azahexadecane-16-yl) benzamide hydrochloride (TU3627-044) was obtained as a yellow solid.

Figure pat00197

Example 38-3: N- (58- (3- amino-4-formyl-phenoxy) -15,55- dioxo-4,7,10,18,21,24,27,30,33,36 , 39,42,45,48,51-pentadecaoxa-14,54-diazaoctapentacontyl-4-((4- (2- (5-amino-8-methylbenzo [f] [1, 7] Synthesis of naphthyridin-2-yl) ethyl) -3-methylphenoxy) methyl) benzamide (X3678-114).

Figure pat00198

A mixture of dPEG-acid F (100 mg), HATU (52.8 mg) and DIEA (97 μl) in 2.0 mL DMF was stirred at room temperature for 30 minutes. Amine D (prepared from 109 mg of compound C) was added and the reaction was stirred at room temperature until completion by monitoring by HPLC. The product was isolated by preparative RP-HPLC. Boc groups of product G (138 mg) from the previous step were removed by treatment with 3 N HCl in methanol and then concentrated to dryness. A mixture of lithium 4- (3-amino-4-formylphenoxy) butanoate (25.2 mg), HATU (38.0 mg) and DIEA (69.7 μl) in 2.0 mL DMF was stirred at room temperature for 30 minutes. Amine H was added and the reaction stirred at room temperature until monitored to completion by HPLC. The product (X3678-114) was isolated by preparative HPLC.

Figure pat00199

Example 38-4: 2- (4-acetyl-3- aminophenoxy ) -N- (3 -nitrobenzyl ) acetamide Synthesis of (TU633-068).

Figure pat00200

Lithium 2- (4-acetyl-3-aminophenoxy) acetate (215 mg), HBTU (379 mg), and 10 mL DMF were combined in 20 mL glass vials and the resulting slurry was stirred at ambient temperature for 1 hour. At this point the reaction mixture became nearly homogeneous. Then 3'-nitrobenzylamine HCl (192 mg) and DIEA (191 μl) were added to the vial and the reaction was stirred at ambient temperature for 30 minutes. The reaction mixture is diluted with EtOAc, water, saturated aq. Continuous washing with NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash chromatography (hexane / EtOAc) to afford the title compound as a yellow solid.

Figure pat00201

Example 38-5: Preparation of 2- (3-amino-4-formyl-phenoxy) -N- (4- nitrobenzyl) acetamide Synthesis of (TU3627-020).

Figure pat00202

Lithium 2- (3-amino-4-formylphenoxy) acetate (44.2 mg) and HBTU (79.6 mg) were placed in a 20 mL glass vial and anhydrous DMF (5 mL) was added. The resulting slurry was stirred at ambient temperature and became homogeneous within 10 minutes. After 15 minutes, DIEA (50 μl) and 4′-nitrobenzylamine HCl (37.7 mg) were added to the reaction mixture and the reaction was stirred at ambient temperature. After 30 minutes, the reaction was confirmed to be complete by LC-MS analysis. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated and the light aq. Citric acid, saturated aq. NaHCO 3 , and saturated aq. Continuous washing with NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to afford the title compound as a tan solid.

Figure pat00203

Example 38-6: Preparation of 2- (3-amino-4-formyl-phenoxy) -N- (2- (2,4- dinitrophenyl) ethyl) acetamide By a similar method as in (TU3627-022).

Figure pat00204

Except for using N 1- (2,4-dinitrophenyl) ethane-1,2-diamine (Oakwood, cat # 015083, 45.2 mg) in place of 4'-nitrobenzylamine HCl in the absence of DIEA And the title compound was prepared in the same manner as TU3627-020. TU3627-022 was obtained as a yellow solid. Rf. 0.15 (SiO 2 , MeOH in 5% DCM).

Figure pat00205

Example 38-7: 4- (3-amino-4-formyl-phenoxy) -N- (2- (2,4- dinitrophenyl) ethyl) Synthesis of butane amide (TU3627-088).

Figure pat00206

Lithium 4- (3-amino-4-formylphenoxy) butanoate (50 mg), HBTU (80 mg) and DMF (2 mL) were combined in 20 mL glass vials and stirred at ambient temperature. After 30 minutes, N 1- (2,4-dinitrophenyl) ethane-1,2-diamine (Oakwood, 45 mg) was added in one portion and the reaction stirred at ambient temperature overnight. The reaction mixture was diluted with EtOAc and the pale aq. Citric acid, water, saturated aq. NaHCO 3 , water, and saturated aq. Continuous washing with NaCl, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to afford the title compound as a yellow solid. Rf: 0.18 (SiO 2 , EtOAc).

Figure pat00207

Example 38-8: 4- (3-amino-4-formyl-phenoxy) -N- (4-nitrobenzyl) butane amide Synthesis of (3793-001).

Figure pat00208

HATU (114.1 mg) was added to a 1 mL DMF solution of lithium 4- (3-amino-4-formylphenoxy) -butanoate (68.7 mg) and the reaction was shaken at room temperature for 1 hour. The resulting solution was then added to a 1 mL DMF solution of 4-nitrobenzylamine HCl salt (56.6 mg) and triethylamine (84 μl), another 1 mL DMF to aid transfer. The reaction was stirred at room temperature for 2 hours. Upon completion, the reaction mixture was partitioned between 4 mL of 10% NaCl (aq) and 8 mL of EtOAc. The phases were separated and the aqueous layer was extracted again with 8 mL EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to afford a crude orange oil. The crude oil was purified by silica gel flash column chromatography with a gradient elution of 0-10% MeOH / DCM to afford the desired product as a light yellow solid.

Figure pat00209

Example 38-9: Synthesis of 3- (4-acetyl-3 -aminophenyl ) -2- (4- nitrobenzamido ) propanoic acid (X3471-116).

Figure pat00210

1 mL DMF was added to 4-nitrobenzoic acid (50.1 mg), HATU (114 mg) and DIEA (105 μl) and the mixture was stirred for 30 minutes. The resulting solution is then added to 3- (4-acetyl-3-aminophenyl) -2-aminopropanoic acid (66.7 mg, 1.0 equiv) in 1.0 mL DMF and the reaction is stirred at room temperature while monitored by LC-MS It was. The title product was then isolated by preparative HPLC.

Figure pat00211

Example 38-10: 4- (3-amino-4-formyl-phenoxy) butanoyl Synthesis of -AlaGlySerArgSerGly (D- Ala) LysChaValAlaAlaTrpThrLeuLysAla (D -Ala) Gly-OH (3647-104).

Figure pat00212

Fmoc groups of Fmoc-exPADRE CLEAR resin (549.3 mg, 0.1 mmol, purchased from Peptide International Inc.) were removed with 20% piperidine / DMF (8 mL × 3). The resin was washed with DMF (1.5 mL x 5). The resin was then poured into 3 mL DMF of lithium 4- (3-amino-4-formylphenoxy) -butanoate (34.4 mg), HBTU (45.5 mg), HOBt (16.2 mg), and DIEA (52 μl). The solution was treated for 6 hours at room temperature. The resin was washed with DMF and the peptide was cleaved from the resin at room temperature for 2 hours using H 2 O / Me 2 S / TFA (10/10/80 v / v%, 10 mL). The cutting slurry was filtered through glass wool and most of the TFA was removed from the filtrate by evaporation. The residue was neutralized with 1 N NaOH ( aq ) , diluted with acetonitrile and dialyzed using SpectraPro ™ 7 dialysis membrane (MWCO 1000) in 50% MeCN ( aq ) (4L × 10). The solution remaining in the dialysis membrane was lyophilized to give the desired product as a white solid.

Figure pat00213

Example 38-11 Synthesis of 3- (4-acetyl-3 -aminophenyl ) propanoyl- Gly (D- Ala) LysChaValAlaAlaTrpThrLeuLysAla (D-Ala) Gly-OH (3465-143).

Figure pat00214

Fmoc groups of Fmoc-PADRE CLEAR resin (105.3 mg, 0.02 mmol, purchased from Peptide International Inc.) were removed with 20% piperidine / DMF (8 mL × 3). The resin was washed with DMF (1.5 mL x 5). The resin was then coupled with a 0.5 mL DMF solution of lithium 3- (4-acetyl-3-aminophenyl) propanoate (5.1 mg), HBTU (9.1 mg), and HOBt (3.3 mg) at room temperature for 7 hours. I was. The resin was washed with DMF and the peptide was cleaved off the resin for 2 hours using TIPS / H 2 O / TFA (5/55/90 v / v%, 3 mL). The cutting slurry was filtered through glass wool and most of the TFA was removed from the filtrate by evaporation. The residue was washed with hexane (3 mL x 3 ) and dissolved in 50% MeCN ( aq ) . After lyophilization, a crude product was obtained which was then purified by preparative HPLC to give the desired product as a light yellow powder.

Figure pat00215

Example 38-12: Preparation of 6- (4- (3-amino-4-formyl-phenoxy) butane amido) hexyl -5'- * T * C * C * A * T * G * A * C * G Synthesis of * T * T * C * C * T * G * A * C * G * T * T-3 '(*: phosphothioate) (3647-057).

Figure pat00216

HBTU (6.1 mg), HOBt (2.2 mg), and triethylamine (7 μl) were added to a 1 mL DMSO solution of lithium 4- (3-amino-4-formylphenoxy) -butanoate (4.6 mg). Added and the reaction was shaken for 1 h at room temperature. 276 μl aliquots of the resulting solution were then subjected to amino-modified BG1 oligos (12.1 mg, 1.8 μmol, purchased from Integrated DNA Technologies, Inc.) and triethylamine (15 Μl) in 1.2 mL DMSO solution and the reaction was shaken for 2 days at room temperature. The reaction mixture was diluted with water and dialyzed against water (4 L × 10) using Slide-A-Riser ™ (MWCO 3500). The dialyzed solution was lyophilized to give the desired product as a white solid.

Figure pat00217

Example 38-13: Preparation of 6- (3- (4-acetyl-3-amino) propane amido) hexyl -5'- * T * C * G * T * C * G * T * T * T * T * C * G * G * C * G * C * G * C * G * C * C * G-3 '(*: phosphothioate) synthesis of (3597-033).

Figure pat00218

HBTU (5.9 mg) and HOBt (2.1 mg) were added to a 1 mL DMSO solution of lithium 3- (4-acetyl-3-aminophenyl) propanoate (3.3 mg) and the reaction was shaken for 1 hour at room temperature. . A 20 μl aliquot of the resulting solution was then placed in a 0.2 mL DMSO solution of amino-modified BG2 oligo (1.88 mg, 0.26 μmol, purchased from Integrated DNA Technologies, Inc.) and triethylamine (2 μl). Was added and the reaction was shaken for 20 h at room temperature. Applying the resulting mixture to a NAP-25 ™ column equilibrated in H 2 O, and eluted with H 2 O. Every 1 mL fraction was monitored by LC-MS and the fractions containing the desired product were combined and lyophilized to give the desired product as a white solid.

Figure pat00219

Example 38-14: Preparation of 6- (4- (3-amino-4-upon-formyl phenoxy) butane amido) hexyl -5'- * T * C * G * T * C * G * T * T * T Synthesis of * T * C * G * G * C * G * C * G * C * G * C * C * G-3 '(*: phosphothioate) (3597-167).

Figure pat00220

HATU (7.4 mg) was added to a 1 mL DMSO solution of lithium 4- (3-amino-4-formylphenoxy) -butanoate (5.5 mg) and the reaction was shaken at room temperature for 1 hour. A 60 μl aliquot of the resulting solution was then added to a 0.6 mL DMSO solution of amino-modified BG2 oligo (6.9 mg, 1.0 μmol) and triethylamine (7.5 μl) and the reaction was shaken at room temperature for 20 hours. . Then another 60 μl aliquot of the freshly prepared activated ester solution was added to the reaction mixture in the same manner except using HBTU instead of HATU and the reaction was shaken for an additional 2 days. The reaction mixture was separated into three portions. Applied to a NAP-25 ™ column equilibrated in the respective portions of H 2 O and eluted with H 2 O. Every 1 mL fraction was monitored by LC-MS and the fractions containing the desired product were combined and lyophilized to give the desired product as a white solid.

Figure pat00221

Example  39: spin cover ABA  Synthesis of Reagents

Example 39-1: N- (3- amino-4-formylphenyl) -1-hydroxy-3-pyrroline-1-oxyl -2,2,5,5- tetramethyl-3-carboxamide Synthesis of (X3626-112b).

Figure pat00222

2.0 mL DMF was added to 2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid (55.3 mg), HBTU (102 mg) and DIEA (105 μl). After stirring for 30 minutes at room temperature, 2,4-diaminobenzaldehyde (40.8 mg) was added. The mixture was stirred at rt until monitored by HPLC and the reaction was complete. The title product was isolated by preparative HPLC.

Figure pat00223

Example  40: biotin ABA  Synthesis of Reagents

Example 40-1: N- (3- amino-4-formylphenyl) -1- (biotin amido) -3,6,9,12- tetrahydro-oxa-15-pentadecane amide (X3626-140) of synthesis.

Figure pat00224

1.0 mL DMF was added to 2,4-diaminobenzaldehyde (11.5 mg), NHS-dPEG ™ 4 biotin (Quantabiodesign, cat # 10200, 50 mg) and DMAP (10.4 mg). The reaction mixture was stirred at rt until monitored by HPLC and the reaction was complete. The product was isolated by silica gel flash chromatography.

Figure pat00225

Example 40-2: N- (1- (3- amino-4-formyl-phenoxy) -2-oxo -7,10,13- tree oxa-3-aza-hexadecane -16- yl) - Biotin amide Synthesis of (X3626-142).

Figure pat00226

1 mL DMF was added to lithium 2- (3-amino-4-formylphenoxy) acetate (45.3 mg), HBTU (77.8 mg) and DIEA (31 μl) and the mixture was stirred for 30 minutes. The resulting solution is added to Biotin-dPEG ™ 3 -NH 3 + TFA (Quantabiodesign, cat # 10193, 100 mg), DIEA (47 μl) in 1.0 mL DMF and the reaction is stirred at room temperature while monitored by LCMS. It was. The product was isolated by preparative HPLC.

Figure pat00227

Example  41: fluorescent light PEG - ABA  Synthesis of Reagents

Example  41-1: Fluororesin - PEG - ABA  ( X3757 -48).

Figure pat00228

DMF (2.0 mL) was added to NHS-fluorescein A (23.6 mg), amine B (17.6 mg) and DIEA (8.7 μl). The mixture was stirred at rt until monitored by HPLC and A was consumed. Product C was isolated by preparative HPLC. ESI-MS (m / z) 679.72 (M−H + ). The product (C, 7.4 mg) was then dissolved in 3 M HCl (1.0 mL), stirred at room temperature for 5 minutes and then methanol was removed by evaporation. This operation was repeated to remove the Boc group to afford amine D. To amine D was added lithium 4- (3-amino-4-formylphenoxy) butanoate (2.3 mg), HBTU (3.8 mg), DIEA (7.0 μl) and DMF (2 mL) at room temperature. The title product was then isolated by preparative HPLC.

Figure pat00229

Example  42: Oligosaccharides - ABA  Synthesis of Reagents

Example 42-1: 3-Amino-4- formylphenoxybutyrate Synthesis of Gal - Glu -1-amide (3793-050).

Figure pat00230

NaBH 3 CN (94.3 mg) was added to a solution of H 2 O (10 mL) of NH 4 OAc (771 mg) and lactose (180 mg) (pH 7) and the reaction was stirred at 35 ° C. for 7 days. The reaction mixture is lyophilized and the residue is dissolved in H 2 O and then passed through Dowex 1 × 8-400 anion exchange resin (OH-form, 45 g) to give excess BH 3 CN , a by-product thereof. And acetate was removed. The eluate was lyophilized to give the crude product, which was purified by cation exchange chromatography using Dow's 50WX8-400 resin (H + form, 30 g) to afford the desired product (3793-048) as a light yellow powder. It was.

Figure pat00231

Lithium 3-amino-4-formylphenoxybutyrate (6.0 mg) was treated with HBTU (9.9 mg) and DIEA (7.7 μl) in anhydrous DMSO (200 μl) for 1 hour. A solution of DMSO (250 μl) of Gal-Glu-1-amine (3793-048) (7.7 mg) from above was then added to the reaction mixture at room temperature and then stirred for 1 day. The reaction mixture was lyophilized and purified by preparative HPLC-MS eluting with NH 4 OAc to afford the desired product (3793-050) as a yellow powder.

Figure pat00232

Example  43: Phospholipids ABA  Synthesis of Reagents

Example  43-1: DOPE - ABA  ( TU3627 -092) synthesis.

Figure pat00233

Lithium 4- (3-amino-4-formylphenoxy) butanoate (34 mg) and HBTU (57 mg) were placed in a 20 mL glass vial and 2 mL DMF was added. The reaction was stirred at ambient temperature for 30 minutes to be activated. In a separate 20 mL vial was added DOPE (76 mg, 1,2, -dioleoyl-sn-glycero-phosphoethanolamine, NOF Corp.) followed by DIEA (35 μL) and 3 mL DCM. The yellow solution of the activated ester in the first vial was transferred to the second vial and the reaction stirred at ambient temperature. After 24 hours, the entire reaction mixture was applied to a 12 g pre-packed SiO 2 column equilibrated with Solvent A (Solvent A: NEt 3 in 5% DMC, Solvent B: NEt 3 in 5% MeOH) and the column was zero → eluted over 15 minutes with a linear gradient of B in 15% A to give a partially purified product as a light yellow, very viscous oil. This product was purified again by flash chromatography using a 12 g SiO 2 column (Solvent A: NEt 3 in 5% DMC, Solvent B: NEt 3 in 5% MeOH) and a linear gradient of B in 2 → 10% A Eluted over 15 minutes. Fractions containing pure product were concentrated under reduced pressure to give the triethylammonium salt of the title compound. Rf: 0.43 (SiO 2 , MeOH in 10% DCM).

Figure pat00234

Example  44: PCL  Reduction of Coupling Connections

It was found to reduce PCL coupling linkages to prevent dissociation of PCL-based protein conjugates. 54A shows ESI mass spectrometric analysis of hFGF21-Lys150PCL reduced to 1 mM with 20 mM NaCNBH 3 after coupling to 2-ABA. 54B shows the ESI mass spectrometric analysis of the reduced hFGF21-Lys150PCL 2-ABA conjugate after dialysis with 10 mM phosphate buffer (pH 7.5) and incubation at 50 ° C. for 1 day.

55 shows the stability of PCL ligation to PEGylated FGF21 with or without reduction by NaCNBH 3 . FGF21 mutant Arg154PCL was reacted and purified with 30.3 kDa-2-ABA-PEG (TU3627-024; see Example 37-7). Purified FGF21Arg154PCL-30.3 kDa-2-ABA-PEG was reduced to 20 mM NaBH 3 CN for 16 hours (room temperature, pH 7.5, 100 mM protein). Samples were incubated for 16 minutes at 4 ° C, room temperature, 37 ° C and 50 ° C, and 95 ° C. SDS-PAGE gels of the reduced and non-reduced samples are shown in FIG. 55A. In addition, FIG. 55B shows SDS-PAGE gels for non-reduced samples incubated at 4 ° C., room temperature, 37 ° C. and 50 ° C., and 95 ° C. for 60 hours.

Example 45: NMR studies of PCL- A ( Lys - P5C ) and PCL- B (Lys-P2C) covalently modified by 2- ABA .

Reaction of PCL-A (3647-125, Example 36-1) and PCL-B (3793-011, Example 36-2) with 2-aminobenzaldehyde (2-ABA) and resulting PCL-ABA addition The structure of water was studied by standard 1D and 2D nuclear magnetic resonance (NMR) spectroscopy.

For reaction of PCL-A and PCL-B with 2-ABA and subsequent characterization of the product, Bruker Avance equipped with 1 H / 13 C / 19 F / 31 P-QNP-low temperature probe NMR data were obtained at 300 K on a 400 MHz NMR instrument (Brooker Biospin, Billerica, Mass.). 1 H 1D spectra were typically recorded with a scan width of 12 ppm with 16 scans, 5 s relaxation delay, 16384 composite data points. 1 H- 1 H COSY spectra were typically recorded in 4 scans, 256 t 1 experiments, and 1 H- 1 H ROESY spectra were recorded in 8 scans, 512 t 1 experiments. 1 H- 13 C HMBC spectra are typically recorded in 32 scans, 256 t 1 experiments, and 1 H- 13 C HMQC spectra are typically 4 scans, 128 t 1 experiments, 222 ppm on the carbon scale and 12 ppm on the proton scale Or a spectral width of 7.5 ppm.

To characterize the reduced adduct, all spectra were recorded at 300 K on a Bruker Avans 600 MHz instrument equipped with a 1 H / 13 C / 15 N-TXI-cold probe. 1 H spectra were recorded with a search range of 14 ppm, typically 64 scans, relaxation delay 2 s, 16384 complex data points, excitation molded for water molecule inhibition. 1 H- 1 H COSY spectra were recorded using a spectral width of 10 ppm, typically in 16 scan, 1024 t 1 experiments. The 1 H- 13 C HMQC spectra were typically recorded in an 8 scan, 256 t 1 experiment with a spectral width of 160 ppm on the carbon scale and 10 ppm on the proton scale. The 1 H- 13 C HMBC spectra were typically recorded as 88 scans, 256 t 1 experiments at 300K using spectral widths of 180 ppm on the carbon scale and 10 ppm on the proton scale.

The reaction of PCL-A with 2-ABA was monitored as follows: 1.0 mg of PCL-A synthesized as described in Example 36-1 was dissolved in 0.5 mL of 1 × PBS in D 2 O. 10 μl of 10 mM 3- (trimethylsilyl) propionic acid (TSP) in D 2 O was added as an internal standard and to determine concentration by NMR. 3.7 mg of 2-aminobenzaldehyde (2-ABA; purchased from Sigma) was dissolved in 0.5 mL of 1 × PBS in D 2 O and 10 μl of 10 mM TSP. The concentration of both samples was measured by NMR. NMR signals of the starting material (Table 7) were subjected to standard NMR methods, including 1 H 1D, 1 H- 1 H COZY, 1 H- 1 H ROESY, 1 H- 13 C HMBC, and 1 H- 13 C HMQC experiments. Assigned.

Figure pat00235

To initiate the reaction, 325 μl of PCL-A solution was mixed with 175 μl of 2-ABA solution. The resulting reaction mixture was transferred to an NMR tube and contained PCL-A and 2-ABA in approximately 1: 1 molar ratio. The reaction proceeds at room temperature and is obtained periodically at the time point when NMR spectrum is indicated (FIG. 56). While the reaction signal for the starting PCL-A material (dot) and the 2-ABA reactant (asterisk) disappeared quickly and the reaction proceeded to completion, all PCL-A was converted (there was a slight excess of 2-ABA in the sample). ). At the first time point obtained 0.5 hours after mixing, two new species were detected at a ratio of approximately 2: 1 (representative resonances are indicated by arrows). A few species were completely converted to major species after several days.

The final reaction product of PCL-A and 2-ABA was prepared using standard 1D 1 H and 13 C, 2D 1 H- 1 H COSY, 1 H- 1 H ROESY, 1 using samples prepared in D 2 O and d6-DMSO. The characteristics were characterized by H- 13 C HMBC and 1 H- 13 C HMQC NMR spectroscopy. Samples in d6-DMSO were purified by HPLC. The signal assignment in the case of proton and carbon resonance and the correlation observed in the 1 H- 13 C HMBC spectrum in d6-DMSO are summarized in Table 8.

Figure pat00236

The selected pass-through correlation for atom numbering and HMBC is shown in FIG. 57A. In contrast to that observed in the PCL-A starting material, two methylene protons on carbon 17 were observed at two different chemical shift values. These protons also showed a heteronuclear bond pass correlation for carbon 7 in the HMBC spectrum, suggesting that a covalent bond was formed between nitrogen 16 and carbon 7. Protons on carbon 7 were resonant at 5.6 ppm (proton 7 is a signal of the PCL-A / 2-ABA adduct highlighted in FIG. 201) and correlated to carbon 2, 8, 9, 5 and 13. Similarly, protons on carbon 2 showed HMBC correlation for carbon 9. These and all other NMR observations for the two samples characterized in D 2 O and D 6 -DMSO are consistent with the structure in FIG. 57A depicted for the main product of the PCL-A / 2-ABA adduct. do. Thus, overall NMR evidence indicates that the structure of the reaction product between PCL-A and 2-ABA is as follows:

Figure pat00237

It was also intended to characterize the structural features of the minor form observed in the reaction mixture (FIG. 56). The low concentration of minority forms and the slow transition to major forms complicate the analysis. The analysis did not reach conclusions. Both minor and major forms had one proton on carbon 7 and the chemical shifts of both were very similar (indicated by arrows at 5.6 ppm in FIG. 56). In addition, the space-through ROESY contact was similar in both forms. The two methylene protons on carbon 17 were degraded, meaning that they showed the same chemical shift in the case of unreacted PCL-A as well as in the minor form of the PCL-A / 2-ABA adduct. However, the chemical shifts of these protons are distinguished in their main form. In addition, these protons show an HMBC correlation for carbon 7 in both minor and major forms, suggesting a covalent bond between carbon 7 and nitrogen 16. It has been observed that the chemical shift of methylene protons on carbon 17 in the minor form is degraded, which means that the covalent bonds between carbon 7 and nitrogen 16 may be semi-stable, and NMR observations of the minor form indicate chemical exchange between two or more species. May imply that The minor form may be an alternative semi-stable stereoisomer of the main form in chemical exchange with the protonated form of the PCL-A / 2-ABA adduct (FIG. 57B).

In addition, a solution of synthetic PCL-A and 2-ABA in D 2 O was mixed as described above and monitored by NMR to allow the reaction to proceed and complete. The addition of an aliquot of the sodium cyanoborohydride (NaCNBH 3 ) solution in D 2 O to the NMR tube reduced the PCL-A / 2-ABA adduct to the new species. Additional aliquots of NaCNBH 3 were added until the resonance at 5.6 ppm (FIG. 56, arrow), which was characteristic for the PCL-A / 2-ABA adduct, disappeared completely. The NMR sample of the fully reduced PCL-A / 2-ABA adduct was then lyophilized and redissolved in anhydrous D 2 O to reconcentrate the sample and remove water. The final sample was reconstituted in 0.5 mL of anhydrous D 2 O. The reduced form was characterized by standard 2D 1 H- 1 H COZY, 1 H- 13 C HMBC and 1 H- 13 C HMQC NMR spectroscopy. The signal assignment and heteronuclear binding pass correlations of proton and carbon resonances from the 1 H- 13 C HMBC spectrum are summarized in Table 9.

Figure pat00238

The numbering of the atoms is shown in Figure 57C. The key difference in the observed correlation in the main form of the PCL-A / 2-ABA adduct is that there is a lack of heteronuclear bond passing correlation between methylene protons on carbon 17 and carbon 7 and between protons on carbon 2 and carbon 9 There is no binding pass correlation at. These and all other NMR observations are consistent with the following (also in FIG. 57C) structure for the reduced PCL-A / 2-ABA adduct:

Figure pat00239

In addition, the reaction of PCL-B with 2-ABA was studied under the same conditions as for the reaction of PCL-A with 2-ABA. A NMR reaction mixture containing PCL-B synthesized as described in Example 36-2 (as above) dissolved in D 2 O and containing PCL-B and 2-ABA in a molar ratio of approximately 1: 1 Transfer to tube. The signal assignments of the starting materials are listed in Table 10.

Figure pat00240

The reaction was run at room temperature and NMR spectra were obtained periodically at the indicated time points (FIG. 58). In contrast to PCL-A samples, PCL-B did not readily react with 2-ABA. Even after 17 days, a large amount of starting material (star for 2-ABA; dot for PCL-B) still remained, and only a small amount was converted to the new species (arrow). These species could not be further characterized. However, NMR analysis of the two reactions clearly showed that the reactivity of PCL-A with 2-ABA is much greater than that of PCL-B.

Example  46: mEGF Pyrrolysine incorporated into ( Pyl ) And PCL of Derivatization

Incorporation of pyrrolysine (Pyl) and PCL into mEGF is achieved as described in Example 14. E. coli BL21 (DE3) cells were co-transformed with the mutant mEGF Tyr10TAG gene on the pAra-pylSTBCD and pET22b vectors. The resulting mEGF mutant is hereinafter referred to as mEGF-Tyr10PCL / Pyl. ESI-MS analysis revealed that both PCL and PYL were incorporated at position 10 of mEGF (FIG. 59A; expected mass for mEGF Tyr10PCL = 7296 Da; expected mass for mEGF Tyr10Pyl = 7310 Da). This gave a mixture of mEGF proteins incorporating PCL or PYL. In this particular sample, the dominant species was mEGF with PCL incorporated therein.

To demonstrate derivatization of this mixture and to demonstrate that the PYL was modified by the methods provided herein, mEGF Tyr10PCL / for 16 hours at 25 ° C. in 10 × PBS (pH 7.0) and 1% (v / v) DMSO. ABA reagent TU3627-014 (see Example 34-2) was coupled to PYL. The conjugation reaction was initiated by adding 10 μM mEGF Tyr10PCL / PYL and 1 mM TU3627-014. Formation of the protein conjugate was confirmed by electrospray ionization-mass spectrometry (ESI-MS) (FIG. 59B). The ratio between PCL and PYL adducts was similar to that of PCL and PYL in unreacted protein (FIG. 58A), indicating similar reactivity to PCL and Pyl.

The examples and embodiments described herein are for illustrative purposes only and it is understood that various modifications or changes will be suggested to those skilled in the art in that respect, and are included within the scope and spirit of the present application and the appended claims. .

                         SEQUENCE LISTING <110> IRM        Geierstanger, Bernhard   <120> BIOSYNTHETICALLY GENERATED PYRROLINE-CARBOXY-LYSINE AND SITE        SPECIFIC PROTEIN MODIFICATIONS VIA CHEMICAL DERIVATIZATION OF        PYRROLINE-CARBOXY-LYSINE AND PYRROLYSINE RESIDUES <130> PAT053160-WO <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 1365 <212> DNA <213> Methanosarcina mazeii <400> 1 atggataaaa aaccactaaa cactctgata tctgcaaccg ggctctggat gtccaggacc 60 ggaacaattc ataaaataaa acaccacgaa gtctctcgaa gcaaaatcta tattgaaatg 120 gcatgcggag accaccttgt tgtaaacaac tccaggagca gcaggactgc aagagcgctc 180 aggcaccaca aatacaggaa gacctgcaaa cgctgcaggg tttcggatga ggatctcaat 240 aagttcctca caaaggcaaa cgaagaccag acaagcgtaa aagtcaaggt cgtttctgcc 300 cctaccagaa cgaaaaaggc aatgccaaaa tccgttgcga gagccccgaa acctcttgag 360 aatacagaag cggcacaggc tcaaccttct ggatctaaat tttcacctgc gataccggtt 420 tccacccaag agtcagtttc tgtcccggca tctgtttcaa catcaatatc aagcatttct 480 acaggagcaa ctgcatccgc actggtaaaa gggaatacga accccattac atccatgtct 540 gcccctgttc aggcaagtgc ccccgcactt acgaagagcc agactgacag gcttgaagtc 600 ctgttaaacc caaaagatga gatttccctg aattccggca agcctttcag ggagcttgag 660 tccgaattgc tctctcgcag aaaaaaagac ctgcagcaga tctacgcgga agaaagggag 720 aattatctgg ggaaactcga gcgtgaaatt accaggttct ttgtggacag gggttttctg 780 gaaataaaat ccccgatcct gatccctctt gagtatatcg aaaggatggg cattgataat 840 gataccgaac tttcaaaaca gatcttcagg gttgacaaga acttctgcct gagacccatg 900 cttgctccaa acctttacaa ctacctgcgc aagcttgaca gggccctgcc tgatccaata 960 aaaatttttg aaataggccc atgctacaga aaagagtccg acggcaaaga acacctcgaa 1020 gagtttacca tgctgaactt ctgccagatg ggatcgggat gcacacggga aaatcttgaa 1080 agcataatta cggacttcct gaaccacctg ggaattgatt tcaagatcgt aggcgattcc 1140 tgcatggtct atggggatac ccttgatgta atgcacggag acctggaact ttcctctgca 1200 gtagtcggac ccataccgct tgaccgggaa tggggtattg ataaaccctg gataggggca 1260 ggtttcgggc tcgaacgcct tctaaaggtt aaacacgact ttaaaaatat caagagagct 1320 gcaaggtccg agtcttacta taacgggatt tctaccaacc tgtaa 1365 <210> 2 <211> 1050 <212> DNA <213> Methanosarcina mazeii <400> 2 atgatccaga aaatggcaac cgaagaactt gacaggttcg gggagaaaat tattgaaggt 60 tttaaattgt ctgatgatga cctcagggct cttctttctc ttgaattcga agaagagctg 120 gaaaagcttt actatgtagc tagaaaggtc agaaactatt atttcggcaa cagggtgttt 180 cttaactgtt ttatttattt ctcaacttat tgtaaaaacc agtgctcttt ttgctactat 240 aactgtaaaa acgaaattaa ccgctaccgc ctgaccggtg aagaggttaa agagatgtgc 300 aaagccctga aaggtgcagg ctttcacatg atcgacctga caatgggaga ggatccctat 360 tactatgatg accctgaccg cttcgttgaa cttgtcagga cagtaaaaga agaactcggg 420 cttccaataa tgatttctcc gggagttatg gatgacagca ccctcctgaa agccagggaa 480 gaaggagcaa atttctttgc cctttatcag gagacttatg accgcgaact ttatggaaag 540 ctaagggtag gtcagtcctt cgaaggaagg tttaatgccc gcaggtttgc aaaagaacag 600 gggtactgta tagaagacgg cattcttacc ggcgtaggaa atgatatcga atcaactctt 660 atatccctga aggggatgaa agcaaacaat cctgatatgg taagggtaat gacttttctg 720 cctcaggaag gaactccgct tgaaggtttc agcgatagtt caaagctttc ggagctgaaa 780 atcatagcga ttctcaggct catgtttcct gaatgcctga taccggcttc tcttgacctt 840 gaaggcatag acggcatggt gcaccgttta aatgccggag caaatattgt aacctccatc 900 ctcccagatt cacgcctgga aggggttgcc aattacgacc gcggcatgga agagagggac 960 agggacgtta caagcgttgt caaaaggctg aaggttatgg gaatggaacc tgcgccgcag 1020 gctgagtttg agagagtcct ggggtgctaa 1050 <210> 3 <211> 1116 <212> DNA <213> Methanosarcina mazeii <400> 3 atgagagagt cctggggtgc tagcctgaaa acaatatgcc ttataggcgg gaagctgcag 60 ggcttcgagg ctgcatacct atctaagaaa gccggaatga aagtgcttgt aatagacaaa 120 aacccgcagg cgcttataag gaattatgcg gatgagttcc agtgttttaa cataacggaa 180 gagccggaaa aactcgtcgc gatatcaaaa aatgttgatg ccatactgcc ggtaaatgaa 240 aaccttgaat gtatagaatt tctgaattct ataaaagaaa aattctcctg cccggtactt 300 ttcgattttg aagcttacag gatcagcagg gataagagaa aatcaaaaga atacttcgca 360 tccataggaa ccccgacccc tcaggacaaa ccgtcggaac caccttattt tgtaaagcct 420 ccctgcgaaa gcagcagtgt gggagcgaga ataatccatg acaggaaaga gcttaaagag 480 cttgagcccg ggatgctcat agaagaatac gttgaagggg aagtggtctc acttgaggtc 540 ataggggatg gaaataattt tgctgtggta aaggaaaccc ttgtacatat cgatgacacc 600 tatgactgcc atatggtgac ccctctccct ctagaccctt ccttcaggga actatcctac 660 tcccttgcag caaacctgcc cttaaaagga attatggacg tggaagcgat ttccggcccc 720 ctggggttaa aagttattga gatagatgcc cgtttcccga gccagactcc gactgcggtc 780 tattattctt ccgggatcaa cctcatagaa ctcctgttcc gggcttttaa tggaggcata 840 gaagagatca aaactctccc tgaagacagg tactgcattt acgaacatct catgcttgca 900 gaaaatggag tacttatccc tgtgggagaa caggtcctgt ccatgggaaa tgattacggc 960 aattattatg aagaacctgg aatagagatt ttcctgtgca aaggagagaa ccctgtattc 1020 accctggttt tctggggcag agacagggaa gaagctgaag ctagaaaaaa caaagggctt 1080 tcgattctaa aaagccgttt cggagctgct gcataa 1116 <210> 4 <211> 795 <212> DNA <213> Methanosarcina mazeii <400> 4 atggcacttt taaccccaga agacctggaa aatattaaca aacagcttca agaagctgat 60 tctactgtcc gcagagttac agggcttgat ataaaaggta tctgtaaaga tttctacggc 120 acaactccat gctgtgaaaa agtaggtatc gtgcctgtga cctcagggaa cgggatcata 180 gggagctttt ccgaatccct gaatgcaatt gccgggtatt tcgggtttga cagttttatt 240 actgatatgc ctgacgtcag cggatattat gaggcagtaa agaacggagc ccggatcata 300 cttatggcag atgataatac cttccttgcc cacaacctga aaaatggaaa aatcgccaat 360 aaccagccgt gtacaggcat aatttatgct gaaatagctt caagatacct gaaagccgat 420 tccaaagaag tgcttgccgt gggtcttggg aaggttggat ttccgggagc agcccatctc 480 gtacagaaag gcttcaaggt ttacggatat gatgctgaca gaacccttct agaaaaaagc 540 gtttccagcc tcggaattat acctttcaat cccgtcagcc ccgaaggcga caggcaaagg 600 aagttttcca ttattttcga agcaaccccc tgtgcagaca cgattccgga atccgtaatt 660 tcggaaaact gtgtgatttc tacccctggg ataccctgtg caatctcaaa ggagctgcaa 720 aaaaagtgtg gagttgaact tgtaatggaa ccactgggga taggtacagc atcaatgctg 780 tattctgtac tctaa 795 <210> 5 <211> 72 <212> DNA <213> Methanosarcina mazeii <400> 5 ggaaacctga tcatgtagat cgaatggact ctaaatccgt tcagccgggt tagattcccg 60 gggtttccgc ca 72 <210> 6 <211> 223 <212> PRT <213> Homo sapiens <400> 6 Met Lys Thr Phe Ile Leu Leu Leu Trp Val Leu Leu Leu Trp Val Ile 1 5 10 15 Phe Leu Leu Pro Gly Ala Thr Ala Gln Pro Glu Arg Asp Cys Arg Val             20 25 30 Ser Ser Phe Arg Val Lys Glu Asn Phe Asp Lys Ala Arg Phe Ser Gly         35 40 45 Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro Glu Gly Leu Phe Leu Gln     50 55 60 Asp Asn Ile Val Ala Glu Phe Ser Val Asp Glu Thr Gly Gln Met Ser 65 70 75 80 Ala Thr Ala Lys Gly Arg Val Arg Leu Leu Asn Asn Trp Asp Val Cys                 85 90 95 Ala Asp Met Val Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala Lys Phe             100 105 110 Lys Met Lys Tyr Trp Gly Val Ala Ser Phe Leu Gln Lys Gly Asn Asp         115 120 125 Asp His Trp Ile Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val Gln Tyr     130 135 140 Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser Tyr Ser 145 150 155 160 Phe Val Phe Ser Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala Gln Lys                 165 170 175 Ile Val Arg Gln Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln Tyr Arg             180 185 190 Leu Ile Val His Asn Gly Tyr Cys Asp Gly Arg Ser Glu Arg Asn Leu         195 200 205 Leu Asp Tyr Lys Asp Asp Asp Asp Lys His His His His His His     210 215 220 <210> 7 <211> 672 <212> DNA <213> Homo sapiens <400> 7 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 8 <211> 672 <212> DNA <213> Homo sapiens <400> 8 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg taggccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 9 <211> 672 <212> DNA <213> Homo sapiens <400> 9 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctagctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 10 <211> 672 <212> DNA <213> Homo sapiens <400> 10 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactagg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 11 <211> 672 <212> DNA <213> Homo sapiens <400> 11 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtagtg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 12 <211> 672 <212> DNA <213> Homo sapiens <400> 12 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtacta gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 13 <211> 672 <212> DNA <213> Homo sapiens <400> 13 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctagctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 14 <211> 672 <212> DNA <213> Homo sapiens <400> 14 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtag 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 15 <211> 672 <212> DNA <213> Homo sapiens <400> 15 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tagaggctga tcgtccacaa cggttactgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 16 <211> 672 <212> DNA <213> Homo sapiens <400> 16 atgaaaacat tcatactcct gctctgggta ctgctgctct gggttatctt cctgcttccc 60 ggtgccactg ctcagcctga gcgcgactgc cgagtgagca gcttccgagt caaggagaac 120 ttcgacaagg ctcgcttctc tgggacctgg tacgccatgg ccaagaagga ccccgagggc 180 ctctttctgc aggacaacat cgtcgcggag ttctccgtgg acgagaccgg ccagatgagc 240 gccacagcca agggccgagt ccgtcttttg aataactggg acgtgtgcgc agacatggtg 300 ggcaccttca cagacaccga ggaccctgcc aagttcaaga tgaagtactg gggcgtagcc 360 tcctttctcc agaaaggaaa tgatgaccac tggatcgtcg acacagacta cgacacgtat 420 gccgtgcagt actcctgccg cctcctgaac ctcgatggca cctgtgctga cagctactcc 480 ttcgtgtttt cccgggaccc caacggcctg cccccagaag cgcagaagat tgtaaggcag 540 cggcaggagg agctgtgcct ggccaggcag tacaggctga tcgtccacaa cggttagtgc 600 gatggcagat cagaaagaaa ccttttggac tataaagacg atgacgataa gcatcaccat 660 caccatcact aa 672 <210> 17 <211> 10 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (7) (7) <223> Xaa is a PCL residue <400> 17 Tyr Trp Gly Val Ala Ser Xaa Leu Gln Lys 1 5 10 <210> 18 <211> 29 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (7) (7) <223> Xaa is Cyc residue <400> 18 Lys Asp Pro Glu Gly Leu Xaa Leu Gln Asp Asn Ile Val Ala Glu Phe 1 5 10 15 Ser Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala Lys             20 25 <210> 19 <211> 166 <212> PRT <213> Homo sapiens <400> 19 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His             20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe         35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp     50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp                 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu             100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala         115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val     130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg                 165 <210> 20 <211> 166 <212> PRT <213> mouse <400> 20 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Val Thr Met Gly Cys Ala Glu Gly             20 25 30 Pro Arg Leu Ser Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe         35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Glu Glu Gln Ala Ile Glu Val Trp     50 55 60 Gln Gly Leu Ser Leu Leu Ser Glu Ala Ile Leu Gln Ala Gln Ala Leu 65 70 75 80 Leu Ala Asn Ser Ser Gln Pro Pro Glu Thr Leu Gln Leu His Ile Asp                 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Ser Leu Leu Arg Val Leu             100 105 110 Gly Ala Gln Lys Glu Leu Met Ser Pro Pro Asp Thr Thr Pro Pro Ala         115 120 125 Pro Leu Arg Thr Leu Thr Val Asp Thr Phe Cys Lys Leu Phe Arg Val     130 135 140 Tyr Ala Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Val 145 150 155 160 Cys Arg Arg Gly Asp Arg                 165 <210> 21 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 21 Met Gly Asp Ser Lys Ile His His His His His Glu Asn Leu Tyr 1 5 10 15 Phe Gln Gly              <210> 22 <211> 301 <212> PRT <213> Homo sapiens <400> 22 Met Gly Ser Asp Lys Ile His His His His His His Glu Asn Leu Tyr 1 5 10 15 Phe Gln Gly Ser Leu Leu Val Asn Pro Glu Gly Pro Thr Leu Met Arg             20 25 30 Leu Asn Ser Val Gln Ser Ser Glu Arg Pro Leu Phe Leu Val His Pro         35 40 45 Ile Glu Gly Ser Thr Thr Val Phe His Ser Leu Ala Ser Arg Leu Ser     50 55 60 Ile Pro Thr Tyr Gly Leu Gln Cys Thr Arg Ala Ala Pro Leu Asp Ser 65 70 75 80 Ile His Ser Leu Ala Ala Tyr Tyr Ile Asp Cys Ile Arg Gln Val Gln                 85 90 95 Pro Glu Gly Pro Tyr Arg Val Ala Gly Tyr Ser Tyr Gly Ala Cys Val             100 105 110 Ala Phe Glu Met Cys Ser Gln Leu Gln Ala Gln Gln Ser Pro Ala Pro         115 120 125 Thr His Asn Ser Leu Phe Leu Phe Asp Gly Ser Pro Thr Tyr Val Leu     130 135 140 Ala Tyr Thr Gln Ser Tyr Arg Ala Lys Leu Thr Pro Gly Ser Glu Ala 145 150 155 160 Glu Ala Glu Thr Glu Ala Ile Cys Phe Phe Val Gln Gln Phe Thr Asp                 165 170 175 Met Glu His Asn Arg Val Leu Glu Ala Leu Leu Pro Leu Lys Gly Leu             180 185 190 Glu Glu Arg Val Ala Ala Ala Val Asp Leu Ile Ile Lys Ser His Gln         195 200 205 Gly Leu Asp Arg Gln Glu Leu Ser Phe Ala Ala Arg Ser Phe Tyr Tyr     210 215 220 Lys Leu Arg Ala Ala Glu Gln Tyr Thr Pro Lys Ala Lys Tyr His Gly 225 230 235 240 Asn Val Met Leu Leu Arg Ala Lys Thr Gly Gly Ala Tyr Gly Glu Asp                 245 250 255 Leu Gly Ala Asp Tyr Asn Leu Ser Gln Val Cys Asp Gly Lys Val Ser             260 265 270 Val His Val Ile Glu Gly Asp His Arg Thr Leu Leu Glu Gly Ser Gly         275 280 285 Leu Glu Ser Ile Ile Ser Ile Ile His Ser Ser Leu Ala     290 295 300 <210> 23 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 23 Met Gly Ser Ser His His His His His His Leu Glu Val Leu Phe Gln 1 5 10 15 Gly Pro          <210> 24 <211> 125 <212> PRT <213> Homo sapiens <400> 24 Met Gly Ser Ser His His His His His His Leu Glu Val Leu Phe Gln 1 5 10 15 Gly Pro Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr             20 25 30 Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu         35 40 45 Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe     50 55 60 Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly 65 70 75 80 Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro                 85 90 95 Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His             100 105 110 Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu         115 120 125 <210> 25 <211> 196 <212> PRT <213> Homo sapiens <400> 25 Met Gly Ser Ser His His His His His His Ser Ser Gly Glu Asn Leu 1 5 10 15 Tyr Phe Gln Gly Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Val Arg             20 25 30 Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu         35 40 45 Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro     50 55 60 Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile 65 70 75 80 Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala                 85 90 95 Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu             100 105 110 Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly         115 120 125 Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala     130 135 140 Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 145 150 155 160 Leu Pro Glu Pro Pro Gly Ile Leu Pro Pro Gln Pro Pro Asp Val Gly                 165 170 175 Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro             180 185 190 Ser Tyr Ala Ser         195 <210> 26 <211> 173 <212> PRT <213> mouse <400> 26 Met Arg Gly Ser His His His His His His Gly Ser Gly Ile Glu Gly 1 5 10 15 Arg Leu Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His             20 25 30 Val Val Ala Asn His Gln Val Glu Glu Gln Leu Trp Leu Ser Gln Arg         35 40 45 Ala Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu     50 55 60 Val Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln Val Leu Phe 65 70 75 80 Lys Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr Val Ser                 85 90 95 Arg Phe Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu Ser Ala Val             100 105 110 Lys Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro         115 120 125 Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly     130 135 140 Asp Gln Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala 145 150 155 160 Glu Ala Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu                 165 170 <210> 27 <211> 62 <212> PRT <213> mouse <400> 27 Met Asn Ser Tyr Pro Gly Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu 1 5 10 15 Asn Gly Gly Val Cys Met His Ile Glu Ser Leu Asp Ser Tyr Thr Cys             20 25 30 Asn Cys Val Ile Gly Tyr Ser Gly Asp Arg Cys Gln Thr Arg Asp Leu         35 40 45 Arg Trp Trp Glu Leu Arg Leu Glu His His His His His His     50 55 60 <210> 28 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <220> <221> MOD_RES <222> (2) (2) <223> D-domain <220> <221> MOD_RES <222> (4) (4) Cyclohexyl-alanine <220> <221> MOD_RES <222> (4) (4) Cyclohexyl-alanine <220> <221> MOD_RES &Lt; 222 > (13) <223> D-domain <400> 28 Gly Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Xaa Gly 1 5 10 <210> 29 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <220> <221> MOD_RES <222> (7) (7) <223> D-domain <220> <221> MOD_RES (222) (9) .. (9) Cyclohexyl-alanine <220> <221> MOD_RES (222) (9) .. (9) Cyclohexyl-alanine <220> <221> MOD_RES <222> (18). (18) <223> D-domain <400> 29 Ala Gly Ser Arg Ser Gly Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys 1 5 10 15 Ala Xaa Gly              <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic construct <220> <221> misc_feature <222> (1) <223> phosphothioate backbone <400> 30 tccatgacgt tcctgacgtt 20 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthetic construct <220> <221> misc_feature <222> (1) (22) <223> phosphothioate backbone <400> 31 tcgtcgtttt cggcgcgcgc cg 22 <210> 32 <211> 22 <212> PRT <213> mouse <220> <221> MOD_RES &Lt; 222 > (11) <223> PCL <400> 32 Met Asn Ser Tyr Pro Gly Cys Pro Ser Ser Xaa Asp Gly Tyr Cys Leu 1 5 10 15 Asn Gly Gly Val Cys Met             20 <210> 33 <211> 22 <212> PRT <213> mouse <220> <221> MISC_FEATURE &Lt; 222 > (11) <223> pyrrolysine <400> 33 Met Asn Ser Tyr Pro Gly Cys Pro Ser Ser Xaa Asp Gly Tyr Cys Leu 1 5 10 15 Asn Gly Gly Val Cys Met             20 <210> 34 <211> 14 <212> PRT <213> mouse <220> <221> MISC_FEATURE &Lt; 222 > (3) <223> pyrrolysine <400> 34 Asn His Xaa Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg 1 5 10 <210> 35 <211> 14 <212> PRT <213> mouse <220> <221> MOD_RES &Lt; 222 > (3) <223> PCL <400> 35 Asn His Xaa Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg 1 5 10

Claims (12)

  1. &Lt; / RTI >
    (I)
    Figure pat00241

    (Wherein,
    R 1 is H or an amino terminal modification group;
    R 2 is OH or a carboxy terminus modification group;
    n is an integer from 1 to 5000;
    Each AA is independently selected from an amino acid residue, a pyrrolysine amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-1, and a pyrrolysine analog amino acid residue having the structure of Formula B-1, ;
    Figure pat00242

    Wherein R 6 is H or C 1 alkyl
    At least one AA is a pyrrolysine analog amino acid residue having the structure of Formula A-1 or Formula B-1, the amino acid residue of Formula A-1 is a residue of the amino acid of Formula V, the amino acid residue of Formula B-1 Is a residue of an amino acid of formula
    Figure pat00243

    Wherein R 6 is H or C 1 alkyl
    Amino acids of formula (V) or formula (VI) are biosynthetically produced in cells in contact with a growth medium comprising a pylC gene and a pylD gene and comprising a precursor)
  2. The compound of claim 1, wherein R 1 is H, the amino acid of formula V is an amino acid having the structure of formula VII, the amino acid of formula VI is an amino acid having the structure of formula VIII, and the precursor is D-ornithine, D Arginine, (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid or (2S) -2-amino-6-((R) -2,5-diaminopentaneami Fig. 8) Hexanoic acid compound.
    Figure pat00244
    Figure pat00245
  3. The compound of claim 1, wherein R 1 is C 1 alkyl, the amino acid of formula V is an amino acid having the structure of formula IX, and the precursor is D-ornithine, D-arginine, (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid or (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid;
    Amino acid of formula VI is an amino acid having a structure of formula X, precursor is D-ornithine, D-arginine, 2,5-diamino-3-methylpentanoic acid, (2R, 3S) -2,5-dia Mino-3-methylpentanoic acid, (2R, 3R) -2,5-diamino-3-methylpentanoic acid, (2S) -2-amino-6- (2,5-diaminopentaneamido) hexanoic acid Or (2S) -2-amino-6-((R) -2,5-diaminopentaneamido) hexanoic acid.
    Figure pat00246
    Figure pat00247
  4. The cell of claim 1, wherein the cells further comprise a pylS gene and a pylT gene, tRNA and aminoacyl tRNA synthetase, wherein the aminoacyl tRNA synthetase is the gene product of the pylS gene, and tRNA Is a gene product of the pylT gene.
  5. The cell of claim 1, wherein the cell further comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS), wherein the O-RS represents the O-tRNA. A compound for aminoacylating an amino acid of Formula V, Formula VI, Formula VII, Formula VIII, Formula IX or Formula X.
  6. The compound of any one of claims 1-3, wherein the cells are prokaryotic or eukaryotic cells.
  7. The method of claim 6, wherein the cells are Escherichia coli ) cells, mammalian cells, yeast cells, insect cells, CHO cells, HeLa cells, HEK293F cells or sf9 cells.
  8. A method of derivatizing a protein comprising contacting a protein having a structure according to Formula I with a reagent of Formula III or Formula IV below.
    (I)
    Figure pat00248

    (Wherein,
    R 1 is H or an amino terminal modification group;
    R 2 is OH or a carboxy terminus modification group;
    n is an integer from 1 to 5000;
    Each AA is independently selected from an amino acid residue, a pyrrolysine amino acid residue, a pyrrolysine analog amino acid residue having the structure of Formula A-1, and a pyrrolysine analog amino acid residue having the structure of Formula B-1, ;
    Figure pat00249

    R 6 is H or C 1 alkyl;
    At least one AA is a pyrrolysine amino acid residue or a pyrrolysine analog amino acid residue having a structure of Formula A-1 or Formula B-1)
    (III)
    Figure pat00250

    (IV)
    Figure pat00251

    (Wherein,
    R 3, R 5 and each R 4 is H, -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, , Aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;
    A is C 3 -C 8 cycloalkyl, C 3 -C 8 heterocycloalkyl, 5 to 6 membered monocyclic aryl, 5 to 6 membered monocyclic heteroaryl, fused 9 to 10 membered bicyclic ring or fused 13 a to 14-membered tricyclic ring, where A is -OH, -NO 2, halo, C 1 - 8 alkyl, halo-substituted -C 1 - 8 alkyl, hydroxy-substituted -C 1 - 8 alkyl, Optionally substituted with 1 to 5 substituents independently selected from aryl, heteroaryl, heterocycloalkyl or cycloalkyl, and -LX 1 ;
    L is a bond, C 1 - 8 alkylene, halo-substituted -C 1 - 8 alkylene, hydroxy-substituted -C 1 - 8 alkylene, C 2-8 alkenylene group, a halo-substituted -C 2 - 8 alkenylene, hydroxy-substituted 2 -C 8 alkenylene,
    Figure pat00252

    Figure pat00253

    -O (CR 11 R 12 ) k- , -S (CR 11 R 12 ) k- , -S (O) k (CR 11 R 12 ) k- , -O (CR 11 R 12 ) k -NR 11 C (O)-, -O (CR 11 R 12 ) k C (O) NR 11- , -C (O)-, -C (O) (CR 11 R 12 ) k- , -C (S)-, -C (S) (CR 11 R 12 ) k- , -C (O) NR 11- , -NR 11 C (O)-, -NR 11 (CR 11 R 12 ) k- , -CONR 11 (CR 11 R 12 ) k- , -N (R 11 ) CO (CR 11 R 12 ) k- , -C (O) NR 11 (CR 11 R 12 ) k- , -NR 11 C (O) (CR 11 R 12 ) k - is selected from wherein each R 11 and R 12 are independently H, C 1 - 8 alkyl, and, - 8 alkyl, halo-substituted -C 1 - 8 alkyl or hydroxy-substituted -C 1 k is an integer from 1 to 12;
    X 1 is a label, dye, polymer, water soluble polymer, polyalkylene glycol, poly (ethylene glycol), derivative of poly (ethylene glycol), sugar, lipid, photocrosslinker, cytotoxic compound, drug, affinity label, photo affinity Labels, reactive compounds; Resins, peptides, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, PCR probes, antisense polynucleotides, ribo-oligonucleotides, deoxy Ribo-oligonucleotides, phosphorothioate-modified DNA, modified DNA and RNA, peptide nucleic acids, saccharides, disaccharides, oligosaccharides, polysaccharides, water soluble dendrimers, cyclodextrins, biomaterials, nanoparticles, spin labels , Fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that covalently or noncovalently interact with other molecules, photocatalytic moieties, chemiradioactive excitation moieties, ligands, photoisomerization moieties, biotin, biotin analogues Residues containing heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox Active agents, aminothio acids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chromophore groups, chemiluminescent groups, fluorescent moieties, electron dense groups, magnetic groups, intercalating groups, chelating groups, chromophores , Energy transfer agents, biologically active agents, detectable labels, small molecules, inhibitory ribonucleic acids, siRNAs, radionucleotides, neutron-trapping agents, derivatives of biotin, quantum dot (s), nanotransmitters, radiotransmitters, abzymes, enzymes , Activated complex activators, viruses, toxins, adjuvants, TLR2 agonists, TLR4 agonists, TLR7 agonists, TLR9 agonists, TLR8 agonists, T-cell epitopes, phospholipids, LPS-like molecules, keyhole limpet hemoshi Non- (KLH), immunogenic hapten, aglycan, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, mimotope, receptor, reverse micelle, detergent, immune booster , brother Dye, FRET reagent, radiation-imaging probe, different probe spectroscopy, prodrugs, toxins for immunotherapy, a solid support, -CH 2 CH 2 - (OCH 2 CH 2 O) p -OX 2, -O- (CH 2 CH 2 O) p CH 2 CH 2 -X 2 , and are selected from any combination thereof, wherein p is 1 to 10,000, X 2 is H, C 1 - 8 alkyl, or a protecting group being the terminal functional group).
  9. The compound of claim 8, wherein ring A is furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, Pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine, pyrazine , Piperazine, triazine, trithiane, indoliazine, indole, isoindole, indolin, benzofuran, benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinolyzine, quinoline, isoquinoline, Cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine, quinuclidin, carbazole, acridine, phenazine, pentazine, phenoxazine, phenyl, indene, naphthalene, azulene, flu Orene, anthracene, phenanthracene, norborane and adamantin.
  10. 10. The method of claim 9, wherein ring A is selected from phenyl, naphthalene and pyridine.
  11. The method of claim 8, wherein the reagent of Formula IV is
    Figure pat00254

    / RTI &gt;
    (Wherein L and X 1 are the same as described in claim 8)
  12. The method of claim 8, wherein the reagent of Formula IV is
    Figure pat00255

    Figure pat00256

    Figure pat00257

    Wherein exPADRE is a peptide according to SEQ ID NO: 28; PADRE is a peptide according to SEQ ID NO: 29; BG1 is a polynucleotide according to SEQ ID NO: 30; BG2 is a polynucleotide according to SEQ ID NO: 31.
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